Oligomer modified diaromatic substituted compounds

ABSTRACT

The invention relates to (among other things) oligomer modified diaromatic substituted compounds. A compound of the invention, when administered by any of a number of administration routes, exhibits advantages over non-oligomer modified diaromatic substituted compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/580,580, filed Oct. 25, 2012, which is a 35 U.S.C. §371application of International Patent Application No. PCT/US2011/025695,filed Feb. 22, 2011, designating the United States, which claims thebenefit of priority under 35 U.S.C. §119(e) to U.S. Provisional PatentApplication No. 61/306,802, filed Feb. 22, 2010, the disclosures ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention comprises (among other things) chemically modifieddiaromatic substituted compounds that possess certain advantages overdiaromatic substituted compounds lacking the chemical modification. Thechemically modified diaromatic substituted compounds described hereinrelate to and/or have application(s) in (among others) the fields ofdrug discovery, pharmacotherapy, physiology, organic chemistry andpolymer chemistry.

BACKGROUND OF THE INVENTION

According to UNAIDS, about 33.3 million people were living with humanimmunodeficiency virus (HIV) at the end of 2009, up from 26.2 millionpeople in 1999. HIV is the etiological agent of acquiredimmunodeficiency syndrome, AIDS. Since the beginning of the epidemic,more than 60 million people have been infected with HIV and nearly 30million people have died of HIV-related causes. In an effort to decreasethe morbidity and mortality associated with HIV infection, a variety ofpharmacological interventions have been proposed.

On Mar. 20, 1987, the FDA approved the use of the compound, AZT(zidovudine), to treat AIDS patients who present with an initial episodeof Pneumocystis carinii pneumonia, AIDS patients with conditions otherthan Pneumocystis carinii pneumonia or patients infected with the viruswith an absolute CD4 lymphocyte count of less than 200/mm³ in theperipheral blood. AZT is a known inhibitor of viral reversetranscriptase, an enzyme necessary for human immunodeficiency virusreplication. U.S. Pat. No. 4,724,232 claims a method of treating humanssuffering from AIDS utilizing AZT.

As disclosed in U.S. Pat. No. 6,043,248, certain antibiotics andpolyanionic dyes inhibit retroviral reverse transcriptase (an enzymenecessary for HIV replication). In addition, publications have reportedthe ability of various sulfated compounds to inhibit virus replication,including HIV. The use of benzodiazepine derivatives have been describedas being potent and selective inhibitors of HIV replications. SeePauwels et al. (1990) Nature 343:470-474 and Merluzzi et al. (199)Science 250:1411-1413.

Building on some of these early attempts (such as 2′,3′dideoxynucleoside analogues such as AZT) to address HIV infection, mucheffort has been focused providing superior agents. In the case of2′,3′-dideoxynucleosides, ddC and ddI have shown potent activity againstHIV in vitro and have been evaluated in clinical trials. See Drug News &Perspectives 5(3):153-169 (1992). Based on this further research, theFDA has approved ddI for the treatment of HIV-1 infections in adults andpediatrics patients who are intolerant to, or whose health hassignificantly deteriorated while on, AZT treatment.

Additional research has lead to the discovery of non-nucleoside reversetranscriptase inhibitors (or NNRTIs). Like the nucleoside analogs,NNRTIs block HIV's infection of via inhibiting reverse transcriptase.Combination treatment using both a nucleoside analog drug (AZT, ddI,ddC, d4T and 3TC) and non-nucleoside reverse transcriptase inhibitors(NNRTIs) are commonly used.

Despite the success experiences with some of these pharmacologicinterventions, challenges remain. For example, it is known that HIVdevelops resistance to many drugs (some in as few as two to seven weekswhen the drug is used alone). Thus, there remains a need to provideadditional drugs to treat individuals infected with HIV.

Delavirdine, a diaromatic substituted compound is an approved NNRTI thathas been in a number of clinical trials. Its efficacy is lower thanother NNRTIs, and it also has an inconvenient schedule. The risk ofcross-resistance across the NNRTI class, as well as its complex set ofdrug interactions, make delavirdine and other NNRTIs less suitable astherapy for HIV patients. The most common adverse event is moderate tosevere rash, which occurs in up to 20% of patients. Other common adverseevents include fatigue, headache and nausea. Liver toxicity has alsobeen reported.

Therefore, pharmacotherapy with such therapeutic diaromatic substitutedcompounds would be improved if these and/or other adverse or sideeffects associated with their use could be decreased or if theirpharmacology may be improved. Thus, there is a large unmet need fordeveloping novel diaromatic substituted compounds to address not onlythese concerns, but the concern associated with resistance as well.

The present invention seeks to address these and other needs in the art.

SUMMARY OF THE INVENTION

In one or more embodiments of the invention, a compound is provided, thecompound comprising diaromatic substituted compound residue covalentlyattached via a stable or degradable linkage to a water-soluble,non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

wherein:

R₁ is —CH₂— or —CO—;

Z is

R₇ is —N(R₇₋₅)H where R₇₋₅ is C₁-C₆ alkyl, —CH₂-cyclopropyl, or—CH₂—CH₂F;

R₈ is —H or —F;

R₉ is —H or —F;

R₁₀ is —H or —F;

Aryl/Heteroaryl is a substituent of formula

wherein X₁₄ is selected from the group consisting of —H, —CH₂-phenyl,—O—CH₂-phenyl, —O—CH₂—COOH, C₁-C₆ alkyl (e.g., C₁-C₄ alkyl), —F, —Cl,—Br, —O—SO₂—(C₁-C₄ alkyl), —NH—SO₂—(C₁-C₆ alkyl), —NO₂, —NH₂, —N₃,—N═CH—N(C₁-C₆ alkyl)(C₁-C₆ alkyl), —N═C(C₁-C₄ alkyl)-N(C₁-C₆alkyl)(C₁-C₆ alkyl), —NH—CO—X₁₄₋₉ where X₁₄₋₉ is —H, —C₁-C₄ alkyl orphenyl; n₇ is 0 or 1; X₆, when present, is —H, —OH, —(CH₂)—OH,—O—CH₂-phenyl, —CHO, C₁-C₃ alkoxy, —O—SO₂—C₁-C₄ alkyl, —NH—SO₂—C₁-C₄alkyl, —C≡N, —O—(CH₂)₂₋₅—N(X₆₋₃)(X₆₋₄) where X₆₋₃ and X₆₋₄ are both —Hor where X₆₋₃ and X₆₋₄ are taken together with the attached nitrogenatom to form a heterocyclic ring selected from the group consisting of1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, N-morpholinyl and1-aziridinyl;

X is a spacer moiety; and

POLY is a water-soluble, non-peptidic oligomer,

and pharmaceutically acceptable salts thereof.

The “diaromatic substituted compound residue” is a compound having astructure of a diaromatic substituted compound that is altered by thepresence of one or more bonds, which bonds serve to attach (eitherdirectly or indirectly) one or more water-soluble, non-peptidicoligomers.

In this regard, any diaromatic substituted compound having anti-HIVactivity can be used as a diaromatic substituted compound moiety.Exemplary diaromatic substituted compound moieties have a structureencompassed by Formula I:

wherein:

R₁ is —CH₂— or —CO—;

Z is

R₇ is —N(R₇₋₅)H where R₇₋₅ is C₁-C₆ alkyl, —CH₂-cyclopropyl, or—CH₂—CH₂F;

R₈ is —H or —F;

R₉ is —H or —F;

R₁₀ is —H or —F; and

Aryl/Heteroaryl is a substituent of formula

wherein X₁₄ is selected from the group consisting of —H, —CH₂-phenyl,—O—CH₂-phenyl, —O—CH₂—COOH, C₁-C₆ alkyl (e.g., C₁-C₄ alkyl), —F, —Cl,—Br, —O—SO₂—(C₁-C₄ alkyl), —NH—SO₂—(C₁-C₆ alkyl), —NO₂, —NH₂, —N₃,—N═CH—N(C₁-C₆ alkyl)(C₁-C₆ alkyl), —N═C(C₁-C₄ alkyl)-N(C₁-C₆alkyl)(C₁-C₆ alkyl), —NH—CO—X₁₄₋₉ where X₁₄₋₉ is —H, —C₁-C₄ alkyl orphenyl; n₇ is 0 or 1; X₆, when present, is —H, —OH, —(CH₂)—OH,—O—CH₂-phenyl, —CHO, C₁-C₃ alkoxy, —O—SO₂—C₁-C₄ alkyl, —NH—SO₂—C₁-C₄alkyl, —C≡N, —O—(CH₂)₂₋₅—N(X₆₋₃)(X₆₋₄) where X₆₋₃ and X₆₋₄ are both —Hor where X₆₋₃ and X₆₋₄ are taken together with the attached nitrogenatom to form a heterocyclic ring selected from the group consisting of1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, N-morpholinyl and1-aziridinyl.

Exemplary diaromatic substituted compound moieties also includeN-[2-({4-[3-(propan-2-ylamino)pyridin-2-yl]piperazin-1-yl}carbonyl)-1H-indol-5-yl]methanesulfonamide.

In one or more embodiments of the invention, a composition is provided,the composition comprising a compound comprising a diaromaticsubstituted compound residue covalently attached via a stable ordegradable linkage to a water-soluble, non-peptidic oligomer, andoptionally, a pharmaceutically acceptable excipient.

In one or more embodiments of the invention, a dosage form is provided,the dosage form comprising a compound comprising a diaromaticsubstituted compound residue covalently attached via a stable ordegradable linkage to a water-soluble, non-peptidic oligomer, whereinthe compound is present in a dosage form.

In one or more embodiments of the invention, a method is provided, themethod comprising covalently attaching a water-soluble, non-peptidicoligomer to a diaromatic substituted compound moiety.

In one or more embodiments of the invention, a method is provided, themethod comprising administering a compound to a mammal in need thereof,comprising a diaromatic substituted compound residue covalently attachedvia a stable or degradable linkage to a water-soluble, non-peptidicoligomer.

Additional embodiments of the present conjugates, compositions, methods,and the like will be apparent from the following description, examples,and claims. As can be appreciated from the foregoing and followingdescription, each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present disclosure provided that the features included insuch a combination are not mutually inconsistent. In addition, anyfeature or combination of features may be specifically excluded from anyembodiment of the present invention. Additional aspects and advantagesof the present invention are set forth in the following description andclaims, particularly when considered in conjunction with theaccompanying examples and drawings.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“Water soluble, non-peptidic oligomer” indicates an oligomer that is atleast 35% (by weight) soluble, preferably greater than 70% (by weight),and more preferably greater than 95% (by weight) soluble, in water atroom temperature. Typically, an unfiltered aqueous preparation of a“water-soluble” oligomer transmits at least 75%, more preferably atleast 95%, of the amount of light transmitted by the same solution afterfiltering. It is most preferred, however, that the water-solubleoligomer is at least 95% (by weight) soluble in water or completelysoluble in water. With respect to being “non-peptidic,” an oligomer isnon-peptidic when it has less than 35% (by weight) of diaromaticsubstituted compound residues.

The terms “monomer,” “monomeric subunit” and “monomeric unit” are usedinterchangeably herein and refer to one of the basic structural units ofa polymer or oligomer. In the case of a homo-oligomer, a singlerepeating structural unit forms the oligomer. In the case of aco-oligomer, two or more structural units are repeated—either in apattern or randomly—to form the oligomer. Preferred oligomers used inconnection with present the invention are homo-oligomers. Thewater-soluble, non-peptidic oligomer comprises one or more monomersserially attached to form a chain of monomers. The oligomer can beformed from a single monomer type (i.e., is homo-oligomeric) or two orthree monomer types (i.e., is co-oligomeric).

An “oligomer” is a molecule possessing from about 1 to about 30monomers. Specific oligomers for use in the invention include thosehaving a variety of geometries such as linear, branched, or forked, tobe described in greater detail below.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Unless otherwise indicated, a“PEG oligomer” or an oligoethylene glycol is one in which substantiallyall (preferably all) monomeric subunits are ethylene oxide subunits,though, the oligomer may contain distinct end capping moieties orfunctional groups, e.g., for conjugation. PEG oligomers for use in thepresent invention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. As stated above, for the PEG oligomers, thevariable (n) ranges from about 1 to 30, and the terminal groups andarchitecture of the overall PEG can vary. When PEG further comprises afunctional group, A, for linking to, e.g., a small molecule drug, thefunctional group when covalently attached to a PEG oligomer does notresult in formation of (i) an oxygen-oxygen bond (—O—O—, a peroxidelinkage), or (ii) a nitrogen-oxygen bond (N—O, O—N).

The terms “end-capped” or “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group. Thus, examples ofend-capping moieties include alkoxy (e.g., methoxy, ethoxy andbenzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. In addition, saturated, unsaturated, substituted and unsubstitutedforms of each of the foregoing are envisioned. Moreover, the end-cappinggroup can also be a silane. The end-capping group can alsoadvantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) of interest towhich the polymer is coupled, can be determined by using a suitabledetector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetricmoieties (e.g., dyes), metal ions, radioactive moieties, and the like.Suitable detectors include photometers, films, spectrometers, and thelike. In addition, the end-capping group may contain a targeting moiety.

The term “targeting moiety” is used herein to refer to a molecularstructure that helps the conjugates of the invention to localize to atargeting area, e.g., help enter a cell, or bind a receptor. Preferably,the targeting moiety comprises of vitamin, antibody, antigen, receptor,DNA, RNA, sialyl Lewis X antigen, hyaluronic acid, sugars, cell specificlectins, steroid or steroid derivative, RGD peptide, ligand for a cellsurface receptor, serum component, or combinatorial molecule directedagainst various intra- or extracellular receptors. The targeting moietymay also comprise a lipid or a phospholipid. Exemplary phospholipidsinclude, without limitation, phosphatidylcholines, phospatidylserine,phospatidylinositol, phospatidylglycerol, and phospatidylethanolamine.These lipids may be in the form of micelles or liposomes and the like.The targeting moiety may further comprise a detectable label oralternately a detectable label may serve as a targeting moiety. When theconjugate has a targeting group comprising a detectable label, theamount and/or distribution/location of the polymer and/or the moiety(e.g., active agent) to which the polymer is coupled can be determinedby using a suitable detector. Such labels include, without limitation,fluorescers, chemiluminescers, moieties used in enzyme labeling,colorimetric (e.g., dyes), metal ions, radioactive moieties, goldparticles, quantum dots, and the like.

“Branched,” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more polymers “arms”extending from a branch point.

“Forked,” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more functional groups(typically through one or more atoms) extending from a branch point.

A “branch point” refers to a bifurcation point comprising one or moreatoms at which an oligomer branches or forks from a linear structureinto one or more additional arms.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive,” with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions that are effective to produce a desiredreaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group may vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as ap-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof encompasses protected forms thereof.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively labile bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater may depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include butare not limited to carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides,oligonucleotides, thioesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “stable” linkage or bond refers to a chemical bond that issubstantially stable in water, that is to say, does not undergohydrolysis under physiological conditions to any appreciable extent overan extended period of time. Examples of hydrolytically stable linkagesinclude but are not limited to the following: carbon-carbon bonds (e.g.,in aliphatic chains), ethers, amides, urethanes, amines, and the like.Generally, a stable linkage is one that exhibits a rate of hydrolysis ofless than about 1-2% per day under physiological conditions. Hydrolysisrates of representative chemical bonds can be found in most standardchemistry textbooks.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater, more preferably 97% or greater, still morepreferably 98% or greater, even more preferably 99% or greater, yetstill more preferably 99.9% or greater, with 99.99% or greater beingmost preferred of some given quantity.

“Monodisperse” refers to an oligomer composition wherein substantiallyall of the oligomers in the composition have a well-defined, singlemolecular weight and defined number of monomers, as determined bychromatography or mass spectrometry. Monodisperse oligomer compositionsare in one sense pure, that is, substantially having a single anddefinable number (as a whole number) of monomers rather than a largedistribution. A monodisperse oligomer composition possesses a MW/Mnvalue of 1.0005 or less, and more preferably, a MW/Mn value of 1.0000.By extension, a composition comprised of monodisperse conjugates meansthat substantially all oligomers of all conjugates in the compositionhave a single and definable number (as a whole number) of monomersrather than a large distribution and would possess a MW/Mn value of1.0005, and more preferably, a MW/Mn value of 1.0000 if the oligomerwere not attached to the therapeutic moiety. A composition comprised ofmonodisperse conjugates may, however, include one or more nonconjugatesubstances such as solvents, reagents, excipients, and so forth.

“Bimodal,” in reference to an oligomer composition; refers to anoligomer composition wherein substantially all oligomers in thecomposition have one of two definable and different numbers (as wholenumbers) of monomers rather than a large distribution, and whosedistribution of molecular weights, when plotted as a number fractionversus molecular weight, appears as two separate identifiable peaks.Preferably, for a bimodal oligomer composition as described herein, eachpeak is generally symmetric about its mean, although the size of the twopeaks may differ. Ideally, the polydispersity index of each peak in thebimodal distribution, Mw/Mn, is 1.01 or less, more preferably 1.001 orless, and even more preferably 1.0005 or less, and most preferably aMW/Mn value of 1.0000. By extension, a composition comprised of bimodalconjugates means that substantially all oligomers of all conjugates inthe composition have one of two definable and different numbers (aswhole numbers) of monomers rather than a large distribution and wouldpossess a MW/Mn value of 1.01 or less, more preferably 1.001 or less andeven more preferably 1.0005 or less, and most preferably a MW/Mn valueof 1.0000 if the oligomer were not attached to the therapeutic moiety. Acomposition comprised of bimodal conjugates may, however, include one ormore nonconjugate substances such as solvents, reagents, excipients, andso forth.

A “diaromatic substituted compound” is broadly used herein to refer toan organic, inorganic, or organometallic compound having a molecularweight of less than about 1000 Daltons and having some degree ofactivity as a diaromatic substituted compound therapeutic. diaromaticsubstituted compound activity of a compound may be measured by assaysknown in the art and also as described herein.

A “biological membrane” is any membrane made of cells or tissues thatserves as a barrier to at least some foreign entities or otherwiseundesirable materials. As used herein a “biological membrane” includesthose membranes that are associated with physiological protectivebarriers including, for example: the blood-brain barrier (BBB); theblood-cerebrospinal fluid barrier; the blood-placental barrier; theblood-milk barrier; the blood-testes barrier; and mucosal barriersincluding the vaginal mucosa, urethral mucosa, anal mucosa, buccalmucosa, sublingual mucosa, and rectal mucosa. Unless the context clearlydictates otherwise, the term “biological membrane” does not includethose membranes associated with the middle gastro-intestinal tract(e.g., stomach and small intestines).

A “biological membrane crossing rate,” provides a measure of acompound's ability to cross a biological membrane, such as theblood-brain barrier (“BBB”). A variety of methods may be used to assesstransport of a molecule across any given biological membrane. Methods toassess the biological membrane crossing rate associated with any givenbiological barrier (e.g., the blood-cerebrospinal fluid barrier, theblood-placental barrier, the blood-milk barrier, the intestinal barrier,and so forth), are known, described herein and/or in the relevantliterature, and/or may be determined by one of ordinary skill in theart.

A “reduced rate of metabolism” refers to a measurable reduction in therate of metabolism of a water-soluble oligomer-small molecule drugconjugate as compared to the rate of metabolism of the small moleculedrug not attached to the water-soluble oligomer (i.e., the smallmolecule drug itself) or a reference standard material. In the specialcase of “reduced first pass rate of metabolism,” the same “reduced rateof metabolism” is required except that the small molecule drug (orreference standard material) and the corresponding conjugate areadministered orally. Orally administered drugs are absorbed from thegastro-intestinal tract into the portal circulation and may pass throughthe liver prior to reaching the systemic circulation. Because the liveris the primary site of drug metabolism or biotransformation, asubstantial amount of drug may be metabolized before it ever reaches thesystemic circulation. The degree of first pass metabolism, and thus, anyreduction thereof, may be measured by a number of different approaches.For instance, animal blood samples may be collected at timed intervalsand the plasma or serum analyzed by liquid chromatography/massspectrometry for metabolite levels. Other techniques for measuring a“reduced rate of metabolism” associated with the first pass metabolismand other metabolic processes are known, described herein and/or in therelevant literature, and/or may be determined by one of ordinary skillin the art. Preferably, a conjugate of the invention may provide areduced rate of metabolism reduction satisfying at least one of thefollowing values: at least about 30%; at least about 40%; at least about50%; at least about 60%; at least about 70%; at least about 80%; and atleast about 90%. A compound (such as a small molecule drug or conjugatethereof) that is “orally bioavailable” is one that preferably possessesa bioavailability when administered orally of greater than 25%, andpreferably greater than 70%, where a compound's bioavailability is thefraction of administered drug that reaches the systemic circulation inunmetabolized form.

“Alkyl” refers to a hydrocarbon chain, ranging from about 1 to 20 atomsin length. Such hydrocarbon chains are preferably but not necessarilysaturated and may be branched or straight chain. Exemplary alkyl groupsinclude methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl,2-ethylpropyl, 3-methylpentyl, and the like. As used herein, “alkyl”includes cycloalkyl when three or more carbon atoms are referenced. An“alkenyl” group is an alkyl of 2 to 20 carbon atoms with at least onecarbon-carbon double bond.

The terms “substituted alkyl” or “substituted C_(q-r) alkyl” where q andr are integers identifying the range of carbon atoms contained in thealkyl group, denotes the above alkyl groups that are substituted by one,two or three halo (e.g., F, Cl, Br, I), trifluoromethyl, hydroxy, C₁₋₇alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, and soforth), C₁₋₇ alkoxy, C₁₋₇ acyloxy, C₃₋₇ heterocyclic, amino, phenoxy,nitro, carboxy, acyl, cyano. The substituted alkyl groups may besubstituted once, twice or three times with the same or with differentsubstituents.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl. “Lower alkenyl” refers to a loweralkyl group of 2 to 6 carbon atoms having at least one carbon-carbondouble bond.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),preferably C₁-C₇.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to component that may be included in the compositions ofthe invention causes no significant adverse toxicological effects to apatient.

The term “aryl” means an aromatic group having up to 14 carbon atoms.Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl,naphthalenyl, and the like. “Substituted phenyl” and “substituted aryl”denote a phenyl group and aryl group, respectively, substituted withone, two, three, four or five (e.g. 1-2, 1-3 or 1-4 substituents) chosenfrom halo (F, Cl, Br, I), hydroxy, cyano, nitro, alkyl (e.g., C₁₋₆alkyl), alkoxy (e.g., C₁₋₆ alkoxy), benzyloxy, carboxy, aryl, and soforth.

Chemical moieties are defined and referred to throughout primarily asunivalent chemical moieties (e.g., alkyl, aryl, etc.). Nevertheless,such terms are also used to convey corresponding multivalent moietiesunder the appropriate structural circumstances clear to those skilled inthe art. For example, while an “alkyl” moiety generally refers to amonovalent radical (e.g., CH₃—CH₂—), in certain circumstances a bivalentlinking moiety can be “alkyl,” in which case those skilled in the artwill understand the alkyl to be a divalent radical (e.g., —CH₂—CH₂—),which is equivalent to the term “alkylene.” (Similarly, in circumstancesin which a divalent moiety is required and is stated as being “aryl,”those skilled in the art will understand that the term “aryl” refers tothe corresponding multivalent moiety, arylene). All atoms are understoodto have their normal number of valences for bond formation (i.e., 1 forH, 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 for S, depending onthe oxidation state of the S).

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a water-soluble oligomer-small moleculedrug conjugate present in a composition that is needed to provide adesired level of active agent and/or conjugate in the bloodstream or inthe target tissue. The precise amount may depend upon numerous factors,e.g., the particular active agent, the components and physicalcharacteristics of the composition, intended patient population, patientconsiderations, and may readily be determined by one skilled in the art,based upon the information provided herein and available in the relevantliterature.

A “difunctional” oligomer is an oligomer having two functional groupscontained therein, typically at its termini. When the functional groupsare the same, the oligomer is said to be homodifunctional. When thefunctional groups are different, the oligomer is said to beheterodifunctional.

A basic reactant or an acidic reactant described herein include neutral,charged, and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as described herein, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may but need not necessarily occur, so that the descriptionincludes instances where the circumstance occurs and instances where itdoes not.

As indicated above, the present invention is directed to (among otherthings) a compound comprising a diaromatic substituted compound residuecovalently attached via a stable or degradable linkage to awater-soluble, non-peptidic oligomer.

The “diaromatic substituted compound residue” is a compound having astructure of a diaromatic substituted compound that is altered by thepresence of one or more bonds, which bonds serve to attach (eitherdirectly or indirectly) one or more water-soluble, non-peptidicoligomers. Exemplary diaromatic substituted compounds have a structureencompassed by at least one of the structures defined as Formula I;

wherein:

R₁ is —CH₂— or —CO—;

Z is

R₇ is —N(R₇₋₅)H where R₇₋₅ is C₁-C₆ alkyl, —CH₂-cyclopropyl, or—CH₂—CH₂F;

R₈ is —H or —F;

R₉ is —H or —F;

R₁₀ is —H or —F; and

Aryl/Heteroaryl is a substituent of formula

wherein X₁₄ is selected from the group consisting of —H, —CH₂-phenyl,—O—CH₂-phenyl, —O—CH₂—COOH, C₁-C₆ alkyl (e.g., C₁-C₄ alkyl), —F, —Br,—O—SO₂—(C₁-C₄ alkyl), —NH—SO₂—(C₁-C₆ alkyl), —NO₂, —NH₂, —N₃,—N═CH—N(C₁-C₆ alkyl)(C₁-C₆ alkyl), —N═C(C₁-C₄ alkyl)-N(C₁-C₆alkyl)(C₁-C₆ alkyl), —NH—CO—X₁₄₋₉ where X₁₄₋₉ is —H, —C₁-C₄ alkyl orphenyl; n₇ is 0 or 1; X₆, when present, is —H, —OH, —(CH₂)—OH,—O—CH₂-phenyl, —CHO, C₁-C₃ alkoxy, —O—SO₂—C₁-C₄ alkyl, —NH—SO₂—C₁-C₄alkyl, —C≡N, —O—(CH₂)₂₋₅—N(X₆₋₃)(X₆₋₄) where X₆₋₃ and X₆₋₄ are both —Hor where X₆₋₃ and X₆₋₄ are taken together with the attached nitrogenatom to form a heterocyclic ring selected from the group consisting of1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, N-morpholinyl and1-aziridinyl.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a diaromatic substituted compound residue covalentlyattached via a stable or degradable linkage to a water-soluble,non-peptidic oligomer, wherein the diaromatic substituted compoundresidue is a residue of delavirdine, the chemical or IUPAC name of whichisN-[2-({4-[3-(propan-2-ylamino)pyridin-2-yl]piperazin-1-yl}carbonyl)-1H-indol-5-yl]methanesulfonamide.

In some instances, diaromatic substituted compounds can be obtained fromcommercial sources. In addition, diaromatic substituted compounds can beobtained through chemical synthesis. Examples of diaromatic substitutedcompounds as well as synthetic approaches for preparing diaromaticsubstituted compounds are described in the literature and in, forexample, U.S. Pat. No. 5,563,142. Each of these (and other) diaromaticsubstituted compounds can be covalently attached (either directly orthrough one or more atoms) to a water-soluble, non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

wherein:

R₁ is —CH₂— or —CO—;

Z is

R₇ is —N(R₇₋₅)H where R₇₋₅ is C₁-C₆ alkyl, —CH₂-cyclopropyl, or—CH₂—CH₂F;

R₈ is —H or —F;

R₉ is —H or —F;

R₁₀ is —H or —F;

Aryl/Heteroaryl is a substituent of formula

wherein X₁₄ is selected from the group consisting of —H, —CH₂-phenyl,—O—CH₂-phenyl, —O—CH₂—COOH, C₁-C₆ alkyl (e.g., C₁-C₄ alkyl), —F, —Cl,—Br, —O—SO₂—(C₁-C₄ alkyl), —NH—SO₂—(C₁-C₆ alkyl), —NO₂, —NH₂, —N₃,—N═CH—N(C₁-C₆ alkyl)(C₁-C₆ alkyl), —N═C(C₁-C₄ alkyl)-N(C₁-C₆alkyl)(C₁-C₆ alkyl), —NH—CO—X₁₄₋₉ where X₁₄₋₉ is —H, —C₁-C₄ alkyl orphenyl; n₇ is 0 or 1; X₆, when present, is —H, —OH, —(CH₂)—OH,—O—CH₂-phenyl, —CHO, C₁-C₃ alkoxy, —O—SO₂—C₁-C₄ alkyl, —NH—SO₂—C₁-C₄alkyl, —C≡N, —O—(CH₂)₂₋₅—N(X₆₋₃)(X₆₋₄) where X₆₋₃ and X₆₋₄ are both —Hor where X₆₋₃ and X₆₋₄ are taken together with the attached nitrogenatom to form a heterocyclic ring selected from the group consisting of1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, N-morpholinyl and1-aziridinyl;

X is a spacer moiety; and

POLY is a water-soluble, non-peptidic oligomer,

and pharmaceutically acceptable salts thereof.

Use of discrete oligomers (e.g., from a monodisperse or bimodalcomposition of oligomers, in contrast to relatively impure compositions)to form oligomer-containing compounds may advantageously alter certainproperties associated with the corresponding small molecule drug. Forinstance, a compound of the invention, when administered by any of anumber of suitable administration routes, such as parenteral, oral,transdermal, buccal, pulmonary, or nasal, exhibits reduced penetrationacross the blood-brain barrier. It is preferred that the compounds ofthe invention exhibit slowed, minimal or effectively no crossing of theblood-brain barrier, while still crossing the gastro-intestinal (GI)walls and into the systemic circulation if oral delivery is intended.Moreover, the compounds of the invention maintain a degree ofbioactivity as well as bioavailability in comparison to the bioactivityand bioavailability of the compound free of all oligomers.

With respect to the blood-brain barrier (“BBB”), this barrier restrictsthe transport of drugs from the blood to the brain. This barrierconsists of a continuous layer of unique endothelial cells joined bytight junctions. The cerebral capillaries, which comprise more than 95%of the total surface area of the BBB, represent the principal route forthe entry of most solutes and drugs into the central nervous system.

For compounds whose degree of blood-brain barrier crossing ability isnot readily known, such ability may be determined using a suitableanimal model such as an in situ rat brain perfusion (“RBP”) model asdescribed herein. Briefly, the RBP technique involves cannulation of thecarotid artery followed by perfusion with a compound solution undercontrolled conditions, followed by a wash out phase to remove compoundremaining in the vascular space. (Such analyses may be conducted, forexample, by contract research organizations such as Absorption Systems,Exton, Pa.). In one example of the RBP model, a cannula is placed in theleft carotid artery and the side branches are tied off. A physiologicbuffer containing the analyte (typically but not necessarily at a 5micromolar concentration level) is perfused at a flow rate of about 10mL/minute in a single pass perfusion experiment. After 30 seconds, theperfusion is stopped and the brain vascular contents are washed out withcompound-free buffer for an additional 30 seconds. The brain tissue isthen removed and analyzed for compound concentrations via liquidchromatography with tandem mass spectrometry detection (LC/MS/MS).Alternatively, blood-brain barrier permeability can be estimated basedupon a calculation of the compound's molecular polar surface area(“PSA”), which is defined as the sum of surface contributions of polaratoms (usually oxygens, nitrogens and attached hydrogens) in a molecule.The PSA has been shown to correlate with compound transport propertiessuch as blood-brain barrier transport. Methods for determining acompound's PSA can be found, e.g., in, Ertl, P., et al., J. Med. Chem.2000, 43, 3714-3717; and Kelder, J., et al., Pharm. Res. 1999, 16,1514-1519.

With respect to the blood-brain barrier, the water-soluble, non-peptidicoligomer-small molecule drug conjugate exhibits a blood-brain barriercrossing rate that is reduced as compared to the crossing rate of thesmall molecule drug not attached to the water-soluble, non-peptidicoligomer. Exemplary reductions in blood-brain barrier crossing rates forthe compounds described herein include reductions of: at least about 5%;at least about 10%; at least about 25%; at least about 30%; at leastabout 40%; at least about 50%; at least about 60%; at least about 70%;at least about 80%; or at least about 90%, when compared to theblood-brain barrier crossing rate of the small molecule drug notattached to the water-soluble oligomer. A preferred reduction in theblood-brain barrier crossing rate for a conjugate of the invention is atleast about 20%.

Assays for determining whether a given compound (regardless of whetherthe compound includes a water-soluble, non-peptidic oligomer or not) canact as a diaromatic substituted compound are known and/or may beprepared by one of ordinary skill in the art and are further describedinfra.

Each of these (and other) diaromatic substituted compound moieties canbe covalently attached (either directly or through one or more atoms) toa water-soluble, non-peptidic oligomer.

Exemplary molecular weights of small molecule drugs include molecularweights of: less than about 950; less than about 900; less than about850; less than about 800; less than about 750; less than about 700; lessthan about 650; less than about 600; less than about 550; less thanabout 500; less than about 450; less than about 400; less than about350; and less than about 300 Daltons.

The small molecule drug used in the invention, if chiral, may beobtained from a racemic mixture, or an optically active form, forexample, a single optically active enantiomer, or any combination orratio of enantiomers (i.e., scalemic mixture). In addition, the smallmolecule drug may possess one or more geometric isomers. With respect togeometric isomers, a composition can comprise a single geometric isomeror a mixture of two or more geometric isomers. A small molecule drug foruse in the present invention can be in its customary active form, or maypossess some degree of modification. For example, a small molecule drugmay have a targeting agent, tag, or transporter attached thereto, priorto or after covalent attachment of an oligomer. Alternatively, the smallmolecule drug may possess a lipophilic moiety attached thereto, such asa phospholipid (e.g., distearoylphosphatidylethanolamine or “DSPE,”dipalmitoylphosphatidylethanolamine or “DPPE,” and so forth) or a smallfatty acid. In some instances, however, it is preferred that the smallmolecule drug moiety does not include attachment to a lipophilic moiety.

The diaromatic substituted compound moiety for coupling to awater-soluble, non-peptidic oligomer possesses a free hydroxyl,carboxyl, thio, amino group, or the like (i.e., “handle”) suitable forcovalent attachment to the oligomer. In addition, the diaromaticsubstituted compound moiety may be modified by introduction of areactive group, preferably by conversion of one of its existingfunctional groups to a functional group suitable for formation of astable covalent linkage between the oligomer and the drug.

Accordingly, each oligomer is composed of up to three different monomertypes selected from the group consisting of: alkylene oxide, such asethylene oxide or propylene oxide; olefinic alcohol, such as vinylalcohol, 1-propenol or 2-propenol; vinyl pyrrolidone; hydroxyalkylmethacrylamide or hydroxyalkyl methacrylate, where alkyl is preferablymethyl; α-hydroxy acid, such as lactic acid or glycolic acid;phosphazene, oxazoline, amino acids, carbohydrates such asmonosaccharides, alditol such as mannitol; and N-acryloylmorpholine.Preferred monomer types include alkylene oxide, olefinic alcohol,hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, andα-hydroxy acid. Preferably, each oligomer is, independently, aco-oligomer of two monomer types selected from this group, or, morepreferably, is a homo-oligomer of one monomer type selected from thisgroup.

The two monomer types in a co-oligomer may be of the same monomer type,for example, two alkylene oxides, such as ethylene oxide and propyleneoxide. Preferably, the oligomer is a homo-oligomer of ethylene oxide.Usually, although not necessarily, the terminus (or termini) of theoligomer that is not covalently attached to a small molecule is cappedto render it unreactive. Alternatively, the terminus may include areactive group. When the terminus is a reactive group, the reactivegroup is either selected such that it is unreactive under the conditionsof formation of the final oligomer or during covalent attachment of theoligomer to a small molecule drug, or it is protected as necessary. Onecommon end-functional group is hydroxyl or —OH, particularly foroligoethylene oxides.

The water-soluble, non-peptidic oligomer (e.g., “POLY” in variousstructures provided herein) can have any of a number of differentgeometries. For example, the water-soluble, non-peptidic oligomer can belinear, branched, or forked. Most typically, the water-soluble,non-peptidic oligomer is linear or is branched, for example, having onebranch point. Although much of the discussion herein is focused uponpoly(ethylene oxide) as an illustrative oligomer, the discussion andstructures presented herein can be readily extended to encompass anywater-soluble, non-peptidic oligomers described above.

The molecular weight of the water-soluble, non-peptidic oligomer,excluding the linker portion, is generally relatively low. Exemplaryvalues of the molecular weight of the water-soluble polymer include:below about 1500; below about 1450; below about 1400; below about 1350;below about 1300; below about 1250; below about 1200; below about 1150;below about 1100; below about 1050; below about 1000; below about 950;below about 900; below about 850; below about 800; below about 750;below about 700; below about 650; below about 600; below about 550;below about 500; below about 450; below about 400; below about 350;below about 300; below about 250; below about 200; and below about 100Daltons.

Exemplary ranges of molecular weights of the water-soluble, non-peptidicoligomer (excluding the linker) include: from about 100 to about 1400Daltons; from about 100 to about 1200 Daltons; from about 100 to about800 Daltons; from about 100 to about 500 Daltons; from about 100 toabout 400 Daltons; from about 200 to about 500 Daltons; from about 200to about 400 Daltons; from about 75 to 1000 Daltons; and from about 75to about 750 Daltons.

Preferably, the number of monomers in the water-soluble, non-peptidicoligomer falls within one or more of the following ranges: between about1 and about 30 (inclusive); between about 1 and about 25; between about1 and about 20; between about 1 and about 15; between about 1 and about12; between about 1 and about 10. In certain instances, the number ofmonomers in series in the oligomer (and the corresponding conjugate) isone of 1, 2, 3, 4, 5, 6, 7, or 8. In additional embodiments, theoligomer (and the corresponding conjugate) contains 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 monomers. In yet further embodiments, theoligomer (and the corresponding conjugate) possesses 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 monomers in series. Thus, for example, when thewater-soluble, non-peptidic polymer includes CH₃—(OCH₂CH₂)_(n)—, “n” isan integer that can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, andcan fall within one or more of the following ranges: between about 1 andabout 25; between about 1 and about 20; between about 1 and about 15;between about 1 and about 12; between about 1 and about 10.

When the water-soluble, non-peptidic oligomer has 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 monomers, these values correspond to a methoxy end-cappedoligo(ethylene oxide) having a molecular weights of about 75, 119, 163,207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When theoligomer has 11, 12, 13, 14, or 15 monomers, these values correspond tomethoxy end-capped oligo(ethylene oxide) having molecular weightscorresponding to about 515, 559, 603, 647, and 691 Daltons,respectively.

When the water-soluble, non-peptidic oligomer is attached to thediaromatic substituted compound (in contrast to the step-wise additionof one or more monomers to effectively “grow” the oligomer onto thediaromatic substituted compound), it is preferred that the compositioncontaining an activated form of the water-soluble, non-peptidic oligomerbe monodisperse. In those instances, however, where a bimodalcomposition is employed, the composition will possess a bimodaldistribution centering around any two of the above numbers of monomers.For instance, a bimodal oligomer may have any one of the followingexemplary combinations of monomer subunits: 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, and so forth; 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9,2-10, and so forth; 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, and so forth;4-5, 4-6, 4-7, 4-8, 4-9, 4-10, and so forth; 5-6, 5-7, 5-8, 5-9, 5-10,and so forth; 6-7, 6-8, 6-9, 6-10, and so forth; 7-8, 7-9, 7-10, and soforth; and 8-9, 8-10, and so forth.

In some instances, the composition containing an activated form of thewater-soluble, non-peptidic oligomer will be trimodal or eventetramodal, possessing a range of monomers units as previouslydescribed. Oligomer compositions possessing a well-defined mixture ofoligomers (i.e., being bimodal, trimodal, tetramodal, and so forth) canbe prepared by mixing purified monodisperse oligomers to obtain adesired profile of oligomers (a mixture of two oligomers differing onlyin the number of monomers is bimodal; a mixture of three oligomersdiffering only in the number of monomers is trimodal; a mixture of fouroligomers differing only in the number of monomers is tetramodal), oralternatively, can be obtained from column chromatography of apolydisperse oligomer by recovering the “center cut”, to obtain amixture of oligomers in a desired and defined molecular weight range.

It is preferred that the water-soluble, non-peptidic oligomer isobtained from a composition that is preferably unimolecular ormonodisperse. That is, the oligomers in the composition possess the samediscrete molecular weight value rather than a distribution of molecularweights. Some monodisperse oligomers can be purchased from commercialsources such as those available from Sigma-Aldrich, or alternatively,can be prepared directly from commercially available starting materialssuch as Sigma-Aldrich. Water-soluble, non-peptidic oligomers can beprepared as described in Chen Y., Baker, G. L., J. Org. Chem., 6870-6873(1999), WO 02/098949, and U.S. Patent Application Publication2005/0136031.

When present, the spacer moiety (through which the water-soluble,non-peptidic polymer is attached to the diaromatic substituted compoundmoiety) may be a single bond, a single atom, such as an oxygen atom or asulfur atom, two atoms, or a number of atoms. A spacer moiety istypically but is not necessarily linear in nature. The spacer moiety,“X,” is hydrolytically stable, and is preferably also enzymaticallystable. Preferably, the spacer moiety “X” is one having a chain lengthof less than about 12 atoms, and preferably less than about 10 atoms,and even more preferably less than about 8 atoms and even morepreferably less than about 5 atoms, whereby length is meant the numberof atoms in a single chain, not counting substituents. For instance, aurea linkage such as this, R_(oligomer)—NH—(C═O)—NH—R′_(drug), isconsidered to have a chain length of 3 atoms (—NH—C(O)—NH—). In selectedembodiments, the linkage does not comprise further spacer groups.

In some instances, the spacer moiety “X” comprises an ether, amide,urethane, amine, thioether, urea, or a carbon-carbon bond. Functionalgroups such as those discussed below, and illustrated in the examples,are typically used for forming the linkages. The spacer moiety may lesspreferably also comprise (or be adjacent to or flanked by) other atoms,as described further below.

More specifically, in selected embodiments, a spacer moiety of theinvention, X, may be any of the following: “—” (i.e., a covalent bond,that may be stable or degradable, between the diaromatic substitutedcompound residue and the water-soluble, non-peptidic oligomer), —O—,—NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —CH₂—C(O)O—, —CH₂—OC(O)—,—C(O)O—CH₂—, —OC(O)—CH₂—, C(O)—NH, NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH₂—,—CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—,—O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—; —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂, —CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—N(R⁶)—, R⁶ is H or an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl and substituted aryl. Additionalspacer moieties include, acylamino, acyl, aryloxy, alkylene bridgecontaining between 1 and 5 inclusive carbon atoms, alkylamino,dialkylamino having about 2 to 4 inclusive carbon atoms, piperidino,pyrrolidino, N-(lower alkyl)-2-piperidyl, morpholino, 1-piperizinyl,4-(lower alkyl)-1-piperizinyl, 4-(hydroxyl-lower alkyl)-1-piperizinyl,4-(methoxy-lower alkyl)-1-piperizinyl, and guanidine. In some instances,a portion or a functional group of the drug compound may be modified orremoved altogether to facilitate attachment of the oligomer. In someinstances, it is preferred that X is not an amide, i.e., —CONR— or—RNCO—).

For purposes of the present invention, however, a group of atoms is notconsidered a linkage when it is immediately adjacent to an oligomersegment, and the group of atoms is the same as a monomer of the oligomersuch that the group would represent a mere extension of the oligomerchain.

The linkage “X” between the water-soluble, non-peptidic oligomer and thesmall molecule is formed by reaction of a functional group on a terminusof the oligomer (or nascent oligomer when it is desired to “grow” theoligomer onto the diaromatic substituted compound) with a correspondingfunctional group within the diaromatic substituted compound.Illustrative reactions are described briefly below. For example, anamino group on an oligomer may be reacted with a carboxylic acid or anactivated carboxylic acid derivative on the small molecule, or viceversa, to produce an amide linkage. Alternatively, reaction of an amineon an oligomer with an activated carbonate (e.g. succinimidyl orbenzotriazolyl carbonate) on the drug, or vice versa, forms a carbamatelinkage. Reaction of an amine on an oligomer with an isocyanate(R—N═C═O) on a drug, or vice versa, forms a urea linkage(R—NH—(C═O)—NH—R′). Further, reaction of an alcohol (alkoxide) group onan oligomer with an alkyl halide, or halide group within a drug, or viceversa, forms an ether linkage. In yet another coupling approach, a smallmolecule having an aldehyde function is coupled to an oligomer aminogroup by reductive amination, resulting in formation of a secondaryamine linkage between the oligomer and the small molecule.

A particularly preferred water-soluble, non-peptidic oligomer is anoligomer bearing an aldehyde functional group. In this regard, theoligomer will have the following structure:CH₃O—(CH₂—CH₂—O)_(p)—(CH₂)_(p)—C(O)H, wherein (n) is one of 1, 2, 3, 4,5, 6, 7, 8, 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and 7. Preferred(n) values include 3, 5 and 7 and preferred (p) values 2, 3 and 4.

The termini of the water-soluble, non-peptidic oligomer not bearing afunctional group may be capped to render it unreactive. When theoligomer includes a further functional group at a terminus other thanthat intended for formation of a conjugate, that group is eitherselected such that it is unreactive under the conditions of formation ofthe linkage “X,” or it is protected during the formation of the linkage“X.”

As stated above, the water-soluble, non-peptidic oligomer includes atleast one functional group prior to conjugation. The functional groupcomprises an electrophilic or nucleophilic group for covalent attachmentto a small molecule, depending upon the reactive group contained withinor introduced into the small molecule. Examples of nucleophilic groupsthat may be present in either the oligomer or the small molecule includehydroxyl, amine, hydrazine (—NHNH₂), hydrazide (—C(O)NHNH₂), and thiol.Preferred nucleophiles include amine, hydrazine, hydrazide, and thiol,particularly amine. Most small molecule drugs for covalent attachment toan oligomer will possess a free hydroxyl, amino, thio, aldehyde, ketone,or carboxyl group.

Examples of electrophilic functional groups that may be present ineither the oligomer or the small molecule include carboxylic acid,carboxylic ester, particularly imide esters, orthoester, carbonate,isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. Morespecific examples of these groups include succinimidyl ester orcarbonate, imidazoyl ester or carbonate, benzotriazole ester orcarbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyldisulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, andtresylate (2,2,2-trifluoroethanesulfonate).

Also included are sulfur analogs of several of these groups, such asthione, thione hydrate, thioketal, 2-thiazolidine thione, etc., as wellas hydrates or protected derivatives of any of the above moieties (e.g.aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal,thioketal, thioacetal).

An “activated derivative” of a carboxylic acid refers to a carboxylicacid derivative that reacts readily with nucleophiles, generally muchmore readily than the underivatized carboxylic acid. Activatedcarboxylic acids include, for example, acid halides (such as acidchlorides), anhydrides, carbonates, and esters. Such esters includeimide esters, of the general form —(CO)O—N[(CO)—]₂; for example,N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Alsopreferred are imidazolyl esters and benzotriazole esters. Particularlypreferred are activated propionic acid or butanoic acid esters, asdescribed in co-owned U.S. Pat. No. 5,672,662. These include groups ofthe form —(CH₂)₂₋₃C(═O)O-Q, where Q is preferably selected fromN-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide,N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole,7-azabenzotriazole, and imidazole.

Other preferred electrophilic groups include succinimidyl carbonate,maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate,p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyldisulfide.

These electrophilic groups are subject to reaction with nucleophiles,e.g., hydroxy, thio, or amino groups, to produce various bond types.Preferred for the present invention are reactions which favor formationof a hydrolytically stable linkage. For example, carboxylic acids andactivated derivatives thereof, which include orthoesters, succinimidylesters, imidazolyl esters, and benzotriazole esters, react with theabove types of nucleophiles to form esters, thioesters, and amides,respectively, of which amides are the most hydrolytically stable.Carbonates, including succinimidyl, imidazolyl, and benzotriazolecarbonates, react with amino groups to form carbamates. Isocyanates(R—N═C═O) react with hydroxyl or amino groups to form, respectively,carbamate (RNH—C(O)—OR′) or urea (RNH—C(O)—NHR′) linkages. Aldehydes,ketones, glyoxals, diones and their hydrates or alcohol adducts (i.e.,aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, andketal) are preferably reacted with amines, followed by reduction of theresulting imine, if desired, to provide an amine linkage (reductiveamination).

Several of the electrophilic functional groups include electrophilicdouble bonds to which nucleophilic groups, such as thiols, can be added,to form, for example, thioether bonds. These groups include maleimides,vinyl sulfones, vinyl pyridine, acrylates, methacrylates, andacrylamides. Other groups comprise leaving groups that can be displacedby a nucleophile; these include chloroethyl sulfone, pyridyl disulfides(which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate,thiosulfonate, and tresylate. Epoxides react by ring opening by anucleophile, to form, for example, an ether or amine bond. Reactionsinvolving complementary reactive groups such as those noted above on theoligomer and the small molecule are utilized to prepare the conjugatesof the invention.

In some instances the diaromatic substituted compound may not have afunctional group suited for conjugation. In this instance, it ispossible to modify (or “functionalize”) the “original” diaromaticsubstituted compound so that it does have a functional group suited forconjugation. For example, if the diaromatic substituted compound has anamide group, but an amine group is desired, it is possible to modify theamide group to an amine group by way of a Hofmann rearrangement, Curtiusrearrangement (once the amide is converted to an azide) or Lossenrearrangement (once amide is concerted to hydroxamide followed bytreatment with tolyene-2-sulfonyl chloride/base).

It is possible to prepare a conjugate of small molecule diaromaticsubstituted compound bearing a carboxyl group wherein the carboxylgroup-bearing small molecule diaromatic substituted compound is coupledto an amino-terminated oligomeric ethylene glycol, to provide aconjugate having an amide group covalently linking the small moleculediaromatic substituted compound to the oligomer. This can be performed,for example, by combining the carboxyl group-bearing small moleculediaromatic substituted compound with the amino-terminated oligomericethylene glycol in the presence of a coupling reagent, (such asdicyclohexylcarbodiimide or “DCC”) in an anhydrous organic solvent.

Further, it is possible to prepare a conjugate of a small moleculediaromatic substituted compound bearing a hydroxyl group wherein thehydroxyl group-bearing small molecule diaromatic substituted compound iscoupled to an oligomeric ethylene glycol halide to result in an ether(—O—) linked small molecule conjugate. This can be performed, forexample, by using sodium hydride to deprotonate the hydroxyl groupfollowed by reaction with a halide-terminated oligomeric ethyleneglycol.

Further, it is possible to prepare a conjugate of a small moleculediaromatic substituted compound moiety bearing a hydroxyl group whereinthe hydroxyl group-bearing small molecule diaromatic substitutedcompound moiety is coupled to an oligomeric ethylene glycol bearing anhaloformate group [e.g., CH₃(OCH₂CH₂)_(n)OC(O)-halo, where halo ischloro, bromo, iodo] to result in a carbonate [—O—C(O)—O—] linked smallmolecule conjugate. This can be performed, for example, by combining adiaromatic substituted compound moiety and an oligomeric ethylene glycolbearing a haloformate group in the presence of a nucleophilic catalyst(such as 4-dimethylaminopyridine or “DMAP”) to thereby result in thecorresponding carbonate-linked conjugate.

In another example, it is possible to prepare a conjugate of a smallmolecule diaromatic substituted compound bearing a ketone group by firstreducing the ketone group to form the corresponding hydroxyl group.Thereafter, the small molecule diaromatic substituted compound nowbearing a hydroxyl group can be coupled as described herein.

In still another instance, it is possible to prepare a conjugate of asmall molecule diaromatic substituted compound bearing an amine group.In one approach, the amine group-bearing small molecule diaromaticsubstituted compound and an aldehyde-bearing oligomer are dissolved in asuitable buffer after which a suitable reducing agent (e.g., NaCNBH₃) isadded. Following reduction, the result is an amine linkage formedbetween the amine group of the amine group-containing small moleculediaromatic substituted compound and the carbonyl carbon of thealdehyde-bearing oligomer.

In another approach for preparing a conjugate of a small moleculediaromatic substituted compound bearing an amine group, a carboxylicacid-bearing oligomer and the amine group-bearing small moleculediaromatic substituted compound are combined, in the presence of acoupling reagent (e.g., DCC). The result is an amide linkage formedbetween the amine group of the amine group-containing small moleculediaromatic substituted compound and the carbonyl of the carboxylicacid-bearing oligomer.

While it is believed that the full scope of the conjugates disclosedherein behave as described, an optimally sized oligomer can beidentified as follows.

First, an oligomer obtained from a monodisperse or bimodal water solubleoligomer is conjugated to the small molecule drug. Preferably, the drugis orally bioavailable, and on its own, exhibits a non-negligibleblood-brain barrier crossing rate. Next, the ability of the conjugate tocross the blood-brain barrier is determined using an appropriate modeland compared to that of the unmodified parent drug. If the results arefavorable, that is to say, if, for example, the rate of crossing issignificantly reduced, then the bioactivity of conjugate is furtherevaluated. Preferably, the compounds according to the invention maintaina significant degree of bioactivity relative to the parent drug, i.e.,greater than about 30% of the bioactivity of the parent drug, or evenmore preferably, greater than about 50% of the bioactivity of the parentdrug.

The above steps are repeated one or more times using oligomers of thesame monomer type but having a different number of subunits and theresults compared.

For each conjugate whose ability to cross the blood-brain barrier isreduced in comparison to the non-conjugated small molecule drug, itsoral bioavailability is then assessed. Based upon these results, that isto say, based upon the comparison of conjugates of oligomers of varyingsize to a given small molecule at a given position or location withinthe small molecule, it is possible to determine the size of the oligomermost effective in providing a conjugate having an optimal balancebetween reduction in biological membrane crossing, oral bioavailability,and bioactivity. The small size of the oligomers makes such screeningsfeasible and allows one to effectively tailor the properties of theresulting conjugate. By making small, incremental changes in oligomersize and utilizing an experimental design approach, one can effectivelyidentify a conjugate having a favorable balance of reduction inbiological membrane crossing rate, bioactivity, and oralbioavailability. In some instances, attachment of an oligomer asdescribed herein is effective to actually increase oral bioavailabilityof the drug.

For example, one of ordinary skill in the art, using routineexperimentation, can determine a best suited molecular size and linkagefor improving oral bioavailability by first preparing a series ofoligomers with different weights and functional groups and thenobtaining the necessary clearance profiles by administering theconjugates to a patient and taking periodic blood and/or urine sampling.Once a series of clearance profiles have been obtained for each testedconjugate, a suitable conjugate can be identified.

Animal models (rodents and dogs) can also be used to study oral drugtransport. In addition, non-in vivo methods include rodent everted gutexcised tissue and Caco-2 cell monolayer tissue-culture models. Thesemodels are useful in predicting oral drug bioavailability.

To determine whether the diaromatic substituted compound or theconjugate of a diaromatic substituted compound and a water-solublenon-peptidic polymer has activity as a diaromatic substituted compoundtherapeutic, it is possible to test such a compound. The diaromaticsubstituted compounds may be tested using available antiviral assaysthat are described here and are known to a person skilled in the art.

The compounds of the invention may be administered per se or in the formof a pharmaceutically acceptable salt, and any reference to thecompounds of the invention herein is intended to includepharmaceutically acceptable salts. If used, a salt of a compound asdescribed herein should be both pharmacologically and pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare the free active compound or pharmaceuticallyacceptable salts thereof and are not excluded from the scope of thisinvention. Such pharmacologically and pharmaceutically acceptable saltscan be prepared by reaction of the compound with an organic or inorganicacid, using standard methods detailed in the literature. Examples ofuseful salts include, but are not limited to, those prepared from thefollowing acids: hydrochloric, hydrobromic, sulfuric, nitric,phosphoric, maleic, acetic, salicyclic, p-toluenesulfonic, tartaric,citric, methanesulfonic, formic, malonic, succinic,naphthalene-2-sulphonic and benzenesulphonic, and the like. Also,pharmaceutically acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium, or calcium salts of acarboxylic acid group.

The compounds of the invention may contain one or more chiral centersand for each chiral center, the invention contemplates each opticalisomer as well as any combination or ratio of or an optically activeform, for example, a single optically active enantiomer, or anycombination or ratio of enantiomers (e.g., scalemic and racemicmixtures). In addition, the small molecule drug may possess one or moregeometric isomers. With respect to geometric isomers, a composition cancomprise a single geometric isomer or a mixture of two or more geometricisomers.

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, maltitol, lactitol, xylitol, sorbitol,myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines, fatty acidsand fatty esters; steroids, such as cholesterol; and chelating agents,such as EDTA, zinc and other such suitable cations.

Pharmaceutically acceptable acids or bases may be present as anexcipient in the preparation. Nonlimiting examples of acids that can beused include those acids selected from the group consisting ofhydrochloric acid, acetic acid, phosphoric acid, citric acid, malicacid, lactic acid, formic acid, trichloroacetic acid, nitric acid,perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, andcombinations thereof. Examples of suitable bases include, withoutlimitation, bases selected from the group consisting of sodiumhydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide,ammonium acetate, potassium acetate, sodium phosphate, potassiumphosphate, sodium citrate, sodium formate, sodium sulfate, potassiumsulfate, potassium fumerate, and combinations thereof.

The amount of the conjugate in the composition will vary depending on anumber of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container. Atherapeutically effective dose can be determined experimentally byrepeated administration of increasing amounts of the conjugate in orderto determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. The optimal amount of any individual excipient isdetermined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, excipients will be present in the composition in anamount of about 1% to about 99% by weight, preferably from about 5%-98%by weight, more preferably from about 15-95% by weight of the excipient,with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsand general teachings regarding pharmaceutical compositions aredescribed in “Remington: The Science & Practice of Pharmacy”, 19^(th)ed., Williams & Williams, (1995), the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H.,Handbook of Pharmaceutical Excipients, 3^(rd) Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions can take any number of forms and theinvention is not limited in this regard. Exemplary preparations are mostpreferably in a form suitable for oral administration such as a tablet,caplet, capsule, gel cap, troche, dispersion, suspension, solution,elixir, syrup, lozenge, transdermal patch, spray, suppository, andpowder.

Oral dosage forms are preferred for those conjugates that are orallyactive, and include tablets, caplets, capsules, gel caps, suspensions,solutions, elixirs, and syrups, and can also comprise a plurality ofgranules, beads, powders or pellets that are optionally encapsulated.Such dosage forms are prepared using conventional methods known to thosein the field of pharmaceutical formulation and described in thepertinent texts.

Tablets and caplets, for example, can be manufactured using standardtablet processing procedures and equipment. Direct compression andgranulation techniques are preferred when preparing tablets or capletscontaining the conjugates described herein. In addition to theconjugate, the tablets and caplets will generally contain inactive,pharmaceutically acceptable carrier materials such as binders,lubricants, disintegrants, fillers, stabilizers, surfactants, coloringagents, flow agents, and the like. Binders are used to impart cohesivequalities to a tablet, and thus ensure that the tablet remains intact.Suitable binder materials include, but are not limited to, starch(including corn starch and pregelatinized starch), gelatin, sugars(including sucrose, glucose, dextrose and lactose), polyethylene glycol,waxes, and natural and synthetic gums, e.g., acacia sodium alginate,polyvinylpyrrolidone, cellulosic polymers (including hydroxypropylcellulose, hydroxypropyl methylcellulose, methyl cellulose,microcrystalline cellulose, ethyl cellulose, hydroxyethylcellulose, andthe like), and Veegum. Lubricants are used to facilitate tabletmanufacture, promoting powder flow and preventing particle capping(i.e., particle breakage) when pressure is relieved. Useful lubricantsare magnesium stearate, calcium stearate, and stearic acid.Disintegrants are used to facilitate disintegration of the tablet, andare generally starches, clays, celluloses, algins, gums, or crosslinkedpolymers. Fillers include, for example, materials such as silicondioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose,and microcrystalline cellulose, as well as soluble materials such asmannitol, urea, sucrose, lactose, dextrose, sodium chloride, andsorbitol. Stabilizers, as well known in the art, are used to inhibit orretard drug decomposition reactions that include, by way of example,oxidative reactions.

Capsules are also preferred oral dosage forms, in which case theconjugate-containing composition can be encapsulated in the form of aliquid or gel (e.g., in the case of a gel cap) or solid (includingparticulates such as granules, beads, powders or pellets). Suitablecapsules include hard and soft capsules, and are generally made ofgelatin, starch, or a cellulosic material. Two-piece hard gelatincapsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form (as alyophilizate or precipitate, which can be in the form of a powder orcake), as well as formulations prepared for injection, which are liquidand require the step of reconstituting the dry form of parenteralformulation. Examples of suitable diluents for reconstituting solidcompositions prior to injection include bacteriostatic water forinjection, dextrose 5% in water, phosphate-buffered saline, Ringer'ssolution, saline, sterile water, deionized water, and combinationsthereof.

In some cases, compositions intended for parenteral administration cantake the form of nonaqueous solutions, suspensions, or emulsions,normally being sterile. Examples of nonaqueous solvents or vehicles arepropylene glycol, polyethylene glycol, vegetable oils, such as olive oiland corn oil, gelatin, and injectable organic esters such as ethyloleate.

The parenteral formulations described herein can also contain adjuvantssuch as preserving, wetting, emulsifying, and dispersing agents. Theformulations are rendered sterile by incorporation of a sterilizingagent, filtration through a bacteria-retaining filter, irradiation, orheat.

The conjugate can also be administered through the skin usingconventional transdermal patch or other transdermal delivery system,wherein the conjugate is contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the conjugate is contained in a layer, or “reservoir,”underlying an upper backing layer. The laminated structure can contain asingle reservoir, or it can contain multiple reservoirs.

The conjugate can also be formulated into a suppository for rectaladministration. With respect to suppositories, the conjugate is mixedwith a suppository base material which is (e.g., an excipient thatremains solid at room temperature but softens, melts or dissolves atbody temperature) such as coca butter (theobroma oil), polyethyleneglycols, glycerinated gelatin, fatty acids, and combinations thereof.Suppositories can be prepared by, for example, performing the followingsteps (not necessarily in the order presented): melting the suppositorybase material to form a melt; incorporating the conjugate (either beforeor after melting of the suppository base material); pouring the meltinto a mold; cooling the melt (e.g., placing the melt-containing mold ina room temperature environment) to thereby form suppositories; andremoving the suppositories from the mold.

In some embodiments of the invention, the compositions comprising theconjugates may further be incorporated into a suitable delivery vehicle.Such delivery vehicles may provide controlled and/or continuous releaseof the conjugates and may also serve as a targeting moiety. Non-limitingexamples of delivery vehicles include, adjuvants, synthetic adjuvants,microcapsules, microparticles, liposomes, and yeast cell wall particles.Yeast cells walls may be variously processed to selectively removeprotein component, glucan, or mannan layers, and are referred to aswhole glucan particles (WGP), yeast beta-glucan mannan particles (YGMP),yeast glucan particles (YGP), Rhodotorula yeast cell particles (YCP).Yeast cells such as S. cerevisiae and Rhodotorula sp. are preferred;however, any yeast cell may be used. These yeast cells exhibit differentproperties in terms of hydrodynamic volume and also differ in the targetorgan where they may release their contents. The methods of manufactureand characterization of these particles are described in U.S. Pat. Nos.5,741,495, 4,810,646, 4,992,540, 5,028,703 and 5,607,677, and U.S.Patent Application Publication Nos. 2005/0281781 and 2008/0044438.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with the conjugate. The method comprisesadministering, generally orally, a therapeutically effective amount ofthe conjugate (preferably provided as part of a pharmaceuticalpreparation). Other modes of administration are also contemplated, suchas pulmonary, nasal, buccal, rectal, sublingual, transdermal, andparenteral. As used herein, the term “parenteral” includes subcutaneous,intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal,and intramuscular injection, as well as infusion injections.

In instances where parenteral administration is utilized, it may benecessary to employ somewhat bigger oligomers than those describedpreviously, with molecular weights ranging from about 500 to 30K Daltons(e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000,5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of the particular conjugate.Those of ordinary skill in the art appreciate which conditions aspecific conjugate can effectively treat. The actual dose to beadministered will vary depend upon the age, weight, and generalcondition of the subject as well as the severity of the condition beingtreated, the judgment of the health care professional, and conjugatebeing administered. Therapeutically effective amounts are known to thoseskilled in the art and/or are described in the pertinent reference textsand literature. Generally, a therapeutically effective amount will rangefrom about 0.001 mg to 1000 mg, preferably in doses from 0.01 mg/day to750 mg/day, and more preferably in doses from 0.10 mg/day to 500 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties. Inthe event of an inconsistency between the teachings of thisspecification and the art incorporated by reference, the meaning of theteachings in this specification shall prevail.

EXPERIMENTAL

It is to be understood that while the invention has been described inconjunction with certain preferred and specific embodiments, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All non-PEG chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated. The preparation ofPEG-mers is described in, for example, U.S. Patent ApplicationPublication No. 2005/0136031.

¹H NMR (nuclear magnetic resonance) data was generated by an NMRspectrometer. A list of certain compounds as well as the source of thecompounds is provided below.

Example 1 Synthesis of mPEG_(n)-O-Delavirdine Conjugates

Synthesis of 2-Chloro-3-isopropylpyridine 2

Acetone (1.1 mL, 14.89 mmol) was added to a stirred solution of3-amino-2-chloropyridine 1 (1.662 g, 12.67 mmol) in 1,2-dichloroethaneat room temperature. And then acetic acid (0.7 mL, 12.22 mmol) wasadded. After about 10 min, NaB(OAc)₃H (3.005 g, 13.47 mmol) was added.The resulting mixture was stirred at room temperature for 24 h. More ofacetone (5.5 ml) and NaB(OAc)₃H (356 mg) and DCE (5 mL) were added. HOAc(0.35 mL) was added. After 2 h, MeOH (5 mL) was added. After overnightat room temperature, water was added to quench the reaction. Sat.aqueous potassium carbonate was added to neutralize the acid. Theorganic phase was separated and the aqueous was extracted withdichloromethane (2×20 mL). The combined organic solution was washed withbrine, dried over Na₂SO₄, concentrated. The residue was separated byflash column chromatography on silica gel, eluting with 5-30%EtOAc/hexane to afford 1.20 g product as colorless oil, as well as0.6326 g of starting material. ¹H-NMR (CDCl₃): 7.669 (dd, J=4.5-5.0 Hzand 1.5 Hz, 1H, Ar—H), 7.075 (dd, J=8.0 Hz and 4.5-5.0 Hz, 1H, Ar—H),6.875 (dd, J=8.0 Hz and 1.5 Hz, 1H, Ar—H), 4.194 (br, 1H, NH)3.644-3.578 (m, 1H, CH), 1.260 (d, J=6.5 Hz, 6H, 2 CH₃). LC-MS: 171.1(MH⁺).

Synthesis of 3-Nitro-2-(Boc-piperazinyl)pyridine 6

1-Boc-piperazine 3 (1.135 g, 5.9 mmol) was dissolved in acetonitrile (20mL) at room temperature, and potassium carbonate (1.02 g, 7.4 mmol) wasadded. The mixture was cooled to 0° C. and 2-chloro-3-nitropyridine 5(0.950 g, 5.93 mmol) was added. The mixture was stirred at 0° C. for 20min, at room temperature for 6 h. The mixture was concentrated to removethe solvent. The residue was mixture with water (5 mL) and brine (15mL). The mixture was extracted with dichloromethane (3×20 mL). Thecombined organic solution was washed with brine (˜40 mL), dried oversodium sulfate, concentrated to afford a yellow residue. The residue waspurified by flash column chromatography on silica gel, eluting with5-40% EtOAc/hexane to afford 1.765 g of product in 97% yield. ¹H-NMR(CDCl₃): 8.342 (dd, J=4.5 Hz and 1.5 Hz, 1H, Ar—H), 8.145 (dd, J=8.0 Hzand 1.50 Hz, 1H, Ar—H), 6.788 (dd, J=8.0 Hz and 4.5 Hz, 1H, Ar—H), 3.565(m, 4H, 2 CH₂) 3.43 (br, 4H, 2 CH₂), 1.477 (s, 9H, 3 CH₃).

Synthesis of 3-Amino-2-(Boc-piperazinyl)pyridine 7

3-Nitro-2-(Boc-piperazine)pyridine 6 (1.765 g, 5.72 mmol) was dissolvedin methanol (15 mL) at room temperature. Then Pd(OH)₂ (1.234 g) andcyclohexene (2.0 mL, 19.52 mmol) were added. The resulting mixture washeated at 70° C. for 2 h. The mixture was concentrated to remove thesolvent. DCM was added and then it was filtered through a pad of silicagel, washed with DCM and 10% MeOH/DCM. The collected solution wasconcentrated and dried under high vacuum to afford a slight pink solidas the product (1.384 g, 87% yield). ¹H-NMR (CDCl₃): 7.794 (dd, J=5.0 Hzand 1.5 Hz, 1H, Ar—H), 6.956 (dd, J=7.5 Hz and 1.5 Hz, 1H, Ar—H), 6.854(dd, J=7.5 Hz and 5.0 Hz, 1H, Ar—H), 3.783 (br, 2H, NH₂), 3.571 (t,J=4.5-5.0 Hz, 4H, 2 CH₂), 3.059 (t, J=4.5-5.0 Hz, 4H, 2 CH₂), 1.482 (s,9H, 3 CH₃). LC-MS: 279.2 (MH⁺). (ref. Romero D. L. et al. J. Med. Chem.37, 999-1014, 1994.)

Synthesis of 2-(Boc-piperazinyl)-3-(isopropylamino)pyridine 4

3-amino-2-(Boc-piperazine)pyridine 7 (3.813 g, about 11.16 mmol, crude)was dissolved in dichloroethane (75 mL). Acetone (1.7 mL, 23.12 mmol)was added, followed by an addition of HOAc (1.0 mL, 17.45 mmol) at roomtemperature. After about 10 min, NaBH(OAc)₃ (7.46 g, 33.44 mmol) wasadded. An additional amount of dichloroethane (25 mL) was added. Theresulting mixture was stirred at room temperature for 64 h. 5% aqueousNaHCO₃ was added to quench the reaction. The organic phase was separatedand the aqueous phase was extracted with dichloromethane (2×50 mL). Thecombined organic solution was washed with brine, dried over anhydrousNa₂SO₄, concentrated. The residue was purified via flash columnchromatography over silica gel with 5-35% ethyl acetate in hexane toafford the product as solid in quantitative yield. ¹H-NMR (CDCl₃): 7.673(dd, J=5.0 Hz and 1.5 Hz, 1H, Ar—H), 6.904 (dd, J=8.0 Hz and 5.0 Hz, 1H,Ar—H), 6.812 (dd, J=8.0 Hz and 1.5 Hz, 1H, Ar—H), 4.150 (d, J=7.5 Hz,1H, NH), 3.569-3.517 (m, 5H, 2CH₂ and CH), 2.997 (t, J=5.0 Hz, 4H, 2CH₂), 1.477 (s, 9H, 3 CH₃), 1.23 (d, J=6.0 Hz, 6H, 2 CH₃). (ref. RomeroD. L. et al. J. Med. Chem. 37, 999-1014, 1994.)

Synthesis of 2-Piperazinyl-3-(isopropylamino)pyridine 8

Trifluoroacetic acid (2.0 mL) was added to a stirred solution of2-(Boc-piperazinyl)-3-(i-propylamino)pyridine 4 (1.426 g, 4.45 mmol) indichloromethane (10 mL) at 0° C. The resulting mixture was stirred at 0°C. for 30 min, at room temperature for 1 h 15 min. Additional quantitiesof TFA (2.0 mL) were added. The mixture was stirred at room temperaturefor another 23 h. Sat. K₂CO₃ solution was slowly added to quench thereaction (gas ↑). Small amount of water was added to help the layersseparation. The organic phase was separated and the aqueous phase wasextracted with dichloromethane (2×25 mL). The combined organic solutionwas washed with brine, dried over anhydrous Na₂SO₄, and concentrated toafford the product in quantitative yield. ¹H-NMR (CDCl₃): 7.681 (dd,J=5.0 Hz and 1.5 Hz, 1H, Ar—H), 6.890 (dd, J=8.0 Hz and 5.0 Hz, 1H,Ar—H), 6.800 (dd, J=8.0 Hz and 1.5 Hz, 1H, Ar—H), 4.170 (d, J=7.5 Hz,1H, NH), 3.567-3.485 (m, 1H, CH), 3.046 (s, 8H, 4 CH₂), 1.983 (br, 1H,NH), 1.234 (d, J=6.0 Hz, 6H, 2 CH₃).

Synthesis of Ethyl 5-O-mPEG₃-indole Carboxylate 10 (n=3)

A mixture of ethyl 5-hydroxyindole carboxylate 9 (264 mg, 1.29 mmol),mPEG₃-OMs (632 mg, 2.61 mmol) and potassium carbonate (552 mg, 3.99mmol) in acetonitrile (15 mL) was stirred at room temperature for 50min, at 70° C. for 19 h. Additional quantities of mPEG₃-OMs (340 mg,1.40 mmol) were added. The mixture was stirred at 70° C. for another 23h. The mixture was concentrated to remove the solvent. The residue wasmixed with water, extracted with DCM (3×15 mL). The combined organicextractions were washed with brine, dried over anhydrous sodium sulfate,and concentrated. The residue was separated by flash columnchromatography on silica gel, eluting with 30-70% EtOAc/Hexane to afford327 mg of product (yield: 72%), as well as staring material (12.7 mg,yield: 4.7%). ¹H-NMR (CDCl₃): 8.762 (br, 1H, NH), 7.300 (dt, J=9.0 Hzand 1.0 Hz, 1H, Ar—H), 7.128 (dd, J=2.0 Hz and 1.0 Hz, 1H, Ar—H), 7.084(m, 1H, Ar—H), 7.027 (dd, J=9.0 Hz and 2.0 Hz, 1H, Ar—H), 4.395 (q,J=7.0 Hz, 2H, CH₂), 4.165 (m, 2H, CH₂), 3.885 (m, 2H, CH₂), 3.771-3.751(m, 2H, CH₂), 3.706-3.687 (m, 2H, CH₂), 3.673-3.654 (m, 2H, CH₂),3.562-3.543 (m, 2H, CH₂), 3.378 (s, 3H, CH₃), 1.406 (t, J=7.0 Hz, 3H,CH₃). LC-MS: 352.2 (MH⁺), 374.2 (MNa⁺).

Synthesis of Ethyl 5-O-mPEG₄-indole Carboxylate 10 (n=4)

A mixture of ethyl 5-hydroxyindole carboxylate 9 (1.0734 g, 5.13 mmol),mPEG₄-OMs (690 mg, 2.41 mmol) and potassium carbonate (3.0361 mg, 21.97mmol) in acetone (40 mL) was stirred 60° C. for 7 h. Additionalquantities of mPEG₄-OMs (897 mg, 3.13 mmol) were added. The mixture wasstirred at 60° C. for another 17 h. More mPEG₄-OMs (756 mg, 2.64 mmol)was added. After 23 h at 60° C., mPEG₄-OMs (438 mg, 1.48 mmol) andacetone (30 mL) were added. The mixture was stirred at 60° C. for twodays. The mixture was cooled to room temperature, filtered. The solidwas washed with DCM. The combined organic solution was concentrated. Theresidue was mixed with water, extracted with DCM (2×30 mL). The combinedorganic extractions were washed with brine, dried over anhydrous sodiumsulfate, concentrated. The residue was separated by flash columnchromatography on silica gel, eluting with 30-90% EtOAc/Hexane to afford1.4612 g of product (yield: 72%). ¹H-NMR (CDCl₃): 8.803 (br, 1H, NH),7.300 (dt, J=9.0 Hz and 1.0 Hz, 1H, Ar—H), 7.123 (dd, J=2.5 Hz and 1.0Hz, 1H, Ar—H), 7.078 (d, J=2.5 Hz, 1H, Ar—H), 7.023 (dd, J=9.0 Hz and2.5 Hz, 1H, Ar—H), 4.391 (q, J=7.0 Hz, 2H, CH₂), 4.160 (m, 2H, CH₂),3.881 (m, 2H, CH₂), 3.754-3.730 (m, 2H, CH₂), 3.702-3.624 (m, 8H, 4CH₂), 3.545-3.527 (m, 2H, CH₂), 3.367 (s, 3H, CH₃), 1.403 (t, J=7.0 Hz,3H, CH₃).

Synthesis of Ethyl 5-O-mPEG₆-indole Carboxylate 10 (n=6)

A mixture of ethyl 5-hydroxyindole carboxylate 9 (1.1334 g, 5.41 mmol),mPEG₆-OMs (1.237 g, 3.3 mmol) and potassium carbonate (3.1465 mg, 3.1465mmol) in acetone (45 mL) was stirred 60° C. for 23 h. Additionalquantities of mPEG₆-OMs (953 mg, 2.55 mmol) were added. The mixture wasstirred at 60° C. for 24.5 h. The mixture was cooled to roomtemperature, filtered. The solid was washed with DCM and acetone. Thecombined organic solution was concentrated. The residue was separated byflash column chromatography on silica gel, eluting with 30-50%EtOAc/Hexane to afford 1.977 g of product (yield: 76%). ¹H-NMR (CDCl₃):8.826 (br, 1H, NH), 7.307 (dt, J=9.0 Hz and 1.0 Hz, 1H, Ar—H), 7.126(dd, J=2.0-2.5 Hz and 1.0 Hz, 1H, Ar—H), 7.080 (d, J=2.0 Hz, 1H, Ar—H),7.028 (dd, J=9.0 Hz and 2.0-2.5 Hz, 1H, Ar—H), 4.393 (q, J=7.0 Hz, 2H,CH₂), 4.165 (t, J=4.5-5.0 Hz, 2H, CH₂), 3.881 (t, J=4.5-5.0 Hz, 2H,CH₂), 3.753-3.728 (m, 2H, CH₂), 3.695-3.626 (m, 16H, 8 CH₂), 3.553-3.535(m, 2H, CH₂), 3.373 (s, 3H, CH₃), 1.406 (t, J=7.0 Hz, 3H, CH₃).

The other ethyl 5-O-mPEG_(n)-indole carboxylate 10 (n=5, 7, 8, 9) couldbe synthesized employing the same procedure as the synthesis of5-O-mPEG₆-indole carboxylate.

Ethyl 5-O-mPEG₅-indole Carboxylate 10 (n=5)

¹H-NMR (CDCl₃): 8.790 (br, 1H, NH), 7.303 (dt, J=9.0 Hz and 1.0 Hz, 1H,Ar—H), 7.126 (dd, J=2.0 Hz and 1.0 Hz, 1H, Ar—H), 7.081 (d, J=2.5 Hz,1H, Ar—H), 7.027 (dd, J=9.0 Hz and 2.5 Hz, 1H, Ar—H), 4.392 (q, J=7.0Hz, 2H, CH₂), 4.164 (d, J=4.5-5.0 Hz, 2H, CH₂), 3.881 (d, J=4.0-4.5 Hz,2H, CH₂), 3.752-3.728 (m, 2H, CH₂), 3.701-3.623 (m, 12H, 6 CH₂),3.549-3.350 (m, 2H, CH₂), 3.371 (s, 3H, CH₃), 1.406 (t, J=7.0 Hz, 3H.CH₃).

Ethyl 5-O-mPEG₇-indole Carboxylate 10 (n=7)

¹H-NMR (CDCl₃): 8.851 (br, 1H, NH), 7.310 (dt, J=9.0 Hz and 1.0 Hz, 1H,Ar—H), 7.126 (dd, J=2.0 Hz and 1.0 Hz, 1H, Ar—H), 7.080 (d, J=2.0 Hz,1H, Ar—H), 7.027 (dd, J=9.0 Hz and 2.5 Hz, 1H, Ar—H), 4.393 (q, J=7.0Hz, 2H, CH₂), 4.165 (t, J=5.0 Hz, 2H, CH₂), 3.882 (t, J=5.0 Hz, 2H,CH₂), 3.752-3.728 (m, 2H, CH₂), 3.701-3.628 (m, 20H, 10 CH₂), 3.372 (s,3H, CH₃), 1.406 (t, J=7.0 Hz, 3H. CH₃).

Ethyl 5-O-mPEG₈-indole Carboxylate 10 (n=8)

¹H-NMR (CDCl₃): 8.892 (br, 1H, NH), 7.311 (dt, J=9.0 Hz and 1.0 Hz, 1H,Ar—H), 7.123 (dd, J=2.0 Hz and 1.0 Hz, 1H, Ar—H), 7.077 (d, J=2.0 Hz,1H, Ar—H), 7.025 (dd, J=9.0 Hz and 2.5 Hz, 1H, Ar—H), 4.391 (q, J=7.0Hz, 2H, CH₂), 4.163 (t, J=5.0 Hz, 2H, CH₂), 3.880 (t, J=5.0 Hz, 2H,CH₂), 3.751-3.726 (m, 2H, CH₂), 3.699-3.616 (m, 24H, 12 CH₂),3.548-3.529 (m, 2H, CH₂), 3.369 (s, 3H, CH₃), 1.404 (t, J=7.0 Hz, 3H.CH₃).

Ethyl 5-O-mPEG₉-indole Carboxylate 10 (n=9)

¹H-NMR (CDCl₃): 8.888 (br, 1H, NH), 7.310 (dt, J=9.0 Hz and 1.0 Hz, 1H,Ar—H), 7.121 (dd, J=2.0 Hz and 1.0 Hz, 1H, Ar—H), 7.076 (d, J=2.5 Hz,1H, Ar—H), 7.023 (dd, J=9.0 Hz and 2.0 Hz, 1H, Ar—H), 4.388 (q, J=7.0Hz, 2H, CH₂), 4.161 (t, J=5.0 Hz, 2H, CH₂), 3.878 (t, J=5.0 Hz, 2H,CH₂), 3.749-3.724 (m, 2H, CH₂), 3.698-3.618 (m, 28H, 14 CH₂),3.545-3.527 (m, 2H, CH₂), 3.368 (s, 3H, CH₃), 1.402 (t, J=7.0 Hz, 3H.CH₃).

5-O-mPEG₃-indole 2-Carboxylic Acid 11 (n=3)

KOH (668 mg, 10.26 mmol) in water (5 mL) was added to a stirred solutionof ethyl 5-O-mPEG₃-indole carboxylate 10 (1.184 g, 3.37 mmol) in dioxane(15 mL). The resulting mixture was stirred at room temperature for 27.5h. The reaction mixture was acidified with 1 N HCl to pH 4.18 andextracted with MeOH/DCM (10/90, 3×30 mL). The organic layers werecombined and washed with brine, dried over Na₂SO₄, and concentratedunder reduced pressure to afford a solid as product (1.0242 g, 94%yield). ¹H-NMR (CDCl₃): 8.997 (br, 1H, NH), 7.297 (dt, J=9.0 Hz and 1.0Hz, 1H, Ar—H), 7.207 (dd, J=2.0 Hz and 1.0 Hz, 1H, Ar—H), 7.055 (d,J=2.0 Hz, 1H, Ar—H), 7.034 (dd, J=9.0 Hz and 2.0 Hz, 1H, Ar—H), 4.157(m, 2H, CH₂), 3.893 (m, 2H, CH₂), 3.784-3.757 (m, 2H, CH₂), 3.724-3.704(m, 2H, CH₂), 3.690-3.671 (m, 2H, CH₂), 3.580-3.560 (m, 2H, CH₂), 3.384(s, 3H, CH₃). LC-MS: 324.2 (MH⁺).

5-O-mPEG₄-indole 2-Carboxylic Acid 11 (n=4)

KOH (872.5 mg, 13.40 mmol) in water (6 mL) was added to a stirredsolution of ethyl 5-O-mPEG₄-indole carboxylate 10 (1.4612 g, 3.695 mmol)in dioxane (25 mL). The resulting mixture was stirred at roomtemperature for 23.5 h. More KOH (794 mg, 12.20 mmol) in water (5 mL)was added. The mixture was stirred at room temperature for 16 h. Thereaction mixture was acidified with 1 N HCl to pH 4.08 and extractedwith MeOH/DCM (10/90, 3×30 mL). The organic layers were combined andwashed with brine, dried over Na₂SO₄, and concentrated under reducedpressure to afford a solid as product (1.3139 g, 97% yield). ¹H-NMR(CDCl₃): 8.846 (br, 1H, NH), 7.307 (d, J=9.0 Hz, 1H, Ar—H), 7.219 (dd,J=2.0 Hz and 0.5 Hz, 1H, Ar—H), 7.073 (d, J=2.0 Hz, 1H, Ar—H), 7.053(dd, J=9.0 Hz and 2.0 Hz, 1H, Ar—H), 4.165 (t, J=5.0 Hz, 2H, CH₂), 3.891(t, J=5.0 Hz, 2H, CH₂), 3.766-3.744 (m, 2H, CH₂), 3.716-3.637 (m, 8H, 4CH₂), 3.556-3.537 (m, 2H, CH₂), 3.373 (s, 3H, CH₃).

5-O-mPEG₆-indole 2-Carboxylic Acid 11 (n=6)

KOH (1.4043 g, 21.57 mmol) in water (10 mL) was added to a stirredsolution of ethyl 5-O-mPEG₆-indole carboxylate 10 (1.977 g, 4.09 mmol)in dioxane (15 mL). The resulting mixture was stirred at roomtemperature for 25 h. Water was added to dilute the mixture. Thereaction mixture was acidified with 1 N HCl to pH 4.64 and extractedwith DCM (3×30 mL). The pH of the aqueous solution was checked andadjusted to pH 4.37, extracted with DCM (3×25 mL). The combined organicsolution was washed with brine, dried over Na₂SO₄, and concentratedunder reduced pressure to afford a solid as product (1.7766 g, 95%yield). ¹H-NMR (CDCl₃): 9.010 (br, 1H, NH), 7.310 (d, J=9.0 Hz, 1H,Ar—H), 7.210 (m, 1H, Ar—H), 7.080 (br, 1H, Ar—H), 7.042 (dd, J=9.0 Hzand 2.0-2.5 Hz, 1H, Ar—H), 4.168 (t, J=4.5-5.0 Hz, 2H, CH₂), 3.880 (t,J=4.5-5.0 Hz, 2H, CH₂), 3.753-3.729 (m, 2H, CH₂), 3.694-3.678 (m, 2H,CH₂), 3.651-3.621 (m, 14H, 7 CH₂), 3.567-3.548 (m, 2H, CH₂), 3.382 (s,3H, CH₃).

The other 5-O-mPEG₆-indole 2-carboxylic acids 11 (n=5, 7, 8, 9) weresynthesized from the corresponding ethyl 5-O-mPEG_(n)-indole carboxylateby the same procedure.

5-O-mPEG₅-indole 2-Carboxylic Acid 11 (n=5)

¹H-NMR (CDCl₃): 8.895 (br, 1H, NH), 7.298 (d, J=9.0 Hz, 1H, Ar—H), 7.201(d, J=1.0 Hz, 1H, Ar—H), 7.061 (br, 1H, Ar—H), 7.036 (dd, J=9.0 Hz and2.0-2.5 Hz, 1H, Ar—H), 4.157 (t, J=4.5-5.0 Hz, 2H, CH₂), 3.880 (t,J=4.5-5.0 Hz, 2H, CH₂), 3.755-3.737 (m, 2H, CH₂), 3.704-3.623 (m, 12H, 6CH₂), 3.553-3.534 (m, 2H, CH₂), 3.371 (s, 3H, CH₃).

5-O-mPEG₇-indole 2-Carboxylic Acid 11 (n=7)

¹H-NMR (CDCl₃): 9.435 (br, 1H, NH), 7.299 (d, J=9.0 Hz, 1H, Ar—H), 7.172(m, 1H, Ar—H), 7.026 (br, 1H, Ar—H), 6.992 (dd, J=9.0 Hz and 2.0 Hz, 1H,Ar—H), 4.125 (t, J=4.5-5.0 Hz, 2H, CH₂), 3.858 (t, J=4.5-5.0 Hz, 2H,CH₂), 3.740-3.722 (m, 2H, CH₂), 3.686-3.611 (m, 20H, 10 CH₂),3.547-3.529 (m, 2H, CH₂), 3.360 (s, 3H, CH₃).

5-O-mPEG₉-indole 2-Carboxylic Acid 11 (n=9)

¹H-NMR (CDCl₃): 9.320 (br, 1H, NH), 7.313 (d, J=9.0 Hz, 1H, Ar—H), 7.183(m, 1H, Ar—H), 7.059 (d, J=2.5 Hz, 1H, Ar—H), 7.013 (dd, J=9.0 Hz and2.0-2.5 Hz, 1H, Ar—H), 4.149 (t, J=4.5-5.0 Hz, 2H, CH₂), 3.868 (t,J=4.5-5.0 Hz, 2H, CH₂), 3.742-3.533 (m, 32H, 16 CH₂), 3.366 (s, 3H,CH₃).

Synthesis of 5-O-mPEG₃-Delavirdine 12 (n=3)

5-O-mPEG₃-indole 2-carboxylic acid 11 (n=3) (425.4 mg, 1.32 mmol) and2-piperazinyl-3-(isopropylamino)pyridine 8 (286 mg, 1.3 mmol) weredissolved in anhydrous THF (15 mL) at room temperature.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.3 mL, 1.64 mmol)was added. The resulting mixture was stirred at room temperature for 5h. 5% aq. NaHCO₃ solution was added to quench the reaction. The mixturewas extracted with DCM (4×20 mL). The organic solution was washed withbrine, dried over Na₂SO₄, concentrated. The residue was purified byflash column chromatography on silica gel, eluting with EtOAc/Hexanes toresult in the product (516.8 mg, yield: 76%) as white solid. ¹H-NMR(CDCl₃): 9.901 (br, 1H, NH), 7.689 (dd, J=5.0 Hz and 1.5 Hz, 1H, Ar—H),7.312 (d, J=9.0 Hz, 1H, Ar—H), 7.043 (d, J=2.0 Hz, 1H, Ar—H), 6.956 (dd,J=9.0 Hz and 2.0 Hz, 1H, Ar—H), 6.928 (dd, J=8.0 Hz and 5.0 Hz, 1H,Ar—H), 6.842 (dd, J=8.0 Hz and 1.5 Hz, 1H, Ar—H), 6.727 (d, J=1.5 Hz,1H, Ar—H), 4.187 (d, J=7.5 Hz, 1H, NH), 4.146 (t, J=5.0 Hz, 2H, CH₂),4.078 (br, 4H, 2 CH₂), 3.866 (t, J=5.0 Hz, 2H, CH₂), 3.755-7.737 (m, 2H,CH₂), 3.369-3.674 (m, 2H, CH₂), 3.660-3.641 (m, 2H, CH₂), 3.592-3.529(m, 3H, CH₂ and CH), 3.361 (s, 3H, CH₃), 3.177 (t, J=5.0 Hz, 4H, 2 CH₂),1.254 (d, J=6.5 Hz, 6H, 2 CH₃). LC-MS: 526.2 (MH⁺).

Synthesis of 5-O-mPEG₄-Delavirdine 12 (n=4)

5-O-mPEG₄-indole 2-carboxylic acid 11 (n=4) (680 mg, 1.85 mmol) and2-piperazinyl-3-(isopropylamino)pyridine 8 (406.5 mg, 1.845 mmol) weredissolved in anhydrous THF (15 mL) at room temperature.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.51 mL, 2.79mmol) was added. The resulting mixture was stirred at room temperaturefor 23 h. 10% aq. NaHCO₃ solution was added to quench the reaction. Themixture was extracted with DCM (4×30 mL). The organic solution waswashed with brine, dried over Na₂SO₄, concentrated. The residue waspurified by flash column chromatography on silica gel, eluting with60-100% EtOAc/Hexanes and EtOAc to result in the product (779 mg, yield:74%) as white solid. ¹H-NMR (CDCl₃): 9.012 (br, 1H, NH), 7.695 (dd,J=4.5 Hz and 1.5 Hz, 1H, Ar—H), 7.315 (d, J=9.0 Hz, 1H, Ar—H), 7.062 (d,J=2.5 Hz, 1H, Ar—H), 6.994 (dd, J=9.0 Hz and 2.5 Hz, 1H, Ar—H), 6.942(dd, J=9.0 Hz and 4.5 Hz, 1H, Ar—H), 6.856 (dd, J=8.0 Hz and 1.0-1.5 Hz,1H, Ar—H), 6.741 (d, J=1.0 Hz, 1H, Ar—H), 4.192 (s, 1H, NH), 4.164 (t,J=5.0 Hz, 2H, CH₂), 4.062 (br, 4H, 2 CH₂), 3.885 (t, J=5.0 Hz, 2H, CH₂),3.760-7.739 (m, 2H, CH₂), 3.710-3.634 (m, 8H, 4 CH₂), 3.591-3.532 (m,3H, CH₂ and CH), 3.371 (s, 3H, CH₃), 3.173 (t, J=5.0 Hz, 4H, 2 CH₂),1.267 (d, J=6.0 Hz, 6H, 2 CH₃). LC-MS: 570.3 (MH⁺).

Synthesis of 5-O-mPEG₅-Delavirdine 12 (n=5)

5-O-mPEG₅-indole 2-carboxylic acid 11 (n=5) (371.8 mg, 0.90 mmol) and2-piperazinyl-3-(isopropylamino)pyridine 8 (196 mg, 0.89 mmol) weredissolved in anhydrous THF (32 mL) at room temperature.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.25 mL, 1.37mmol) was added. The resulting mixture was stirred at room temperaturefor 5 h 45 min. 5% aq. NaHCO₃ solution was added to quench the reaction.The mixture was extracted with DCM (4×20 mL). The organic solution waswashed with brine, dried over Na₂SO₄, concentrated. The residue waspurified by flash column chromatography to result in the product (107.5mg, yield: 20%) as oil. ¹H-NMR (CDCl₃): 8.991 (br, 1H, NH), 7.696 (dd,J=4.5-5.0 Hz and 1.0-1.5 Hz, 1H, Ar—H), 7.307 (d, J=9.0 Hz, 1H, Ar—H),7.053 (d, J=2.5 Hz, 1H, Ar—H), 6.986 (dd, J=9.0 Hz and 2.5 Hz, 1H,Ar—H), 6.932 (dd, J=8.0 Hz and 5.0 Hz, 1H, Ar—H), 6.846 (dd, J=7.0 Hzand 1.0 Hz, 1H, Ar—H), 6.732 (d, J=1.5 Hz, 1H, Ar—H), 4.182 (s, 1H, NH),4.155 (t, J=5.0 Hz, 2H, CH₂), 4.051 (br, 4H, 2 CH₂), 3.874 (t, J=5.0 Hz,2H, CH₂), 3.748-7.736 (m, 2H, CH₂), 3.693-3.617 (m, 12H, 6 CH₂),3.597-3.522 (m, 3H, CH₂ and CH), 3.361 (s, 3H, CH₃), 3.133 (t, J=5.0 Hz,4H, 2 CH₂), 1.258 (d, J=6.5 Hz, 6H, 2 CH₃). LC-MS: 614.3 (MH⁺).

Synthesis of 5-O-mPEG₆-Delavirdine 12 (n=6)

5-O-mPEG₆-indole 2-carboxylic acid 11 (n=6) (554 mg, 1.22 mmol) and2-piperazinyl-3-(isopropylamino)pyridine 8 (268 mg, 1.22 mmol) weredissolved in anhydrous THF (40 mL) at room temperature.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.35 mL, 1.92mmol) was added. The resulting mixture was stirred at room temperaturefor 47.5 h. 5% aq. NaHCO₃ solution was added to quench the reaction. Themixture was extracted with DCM (4×20 mL). The organic solution waswashed with brine, dried over Na₂SO₄, concentrated. The residue waspurified by flash column chromatography to result in the product (252mg, yield: 32%) as oil. ¹H-NMR (CDCl₃): 9.110 (br, 1H, NH), 7.696 (dd,J=4.5 Hz and 1.5 Hz, 1H, Ar—H), 7.318 (d, J=9.0 Hz, 1H, Ar—H), 7.058 (d,J=2.0 Hz, 1H, Ar—H), 6.990 (dd, J=9.0 Hz and 2.5 Hz, 1H, Ar—H), 6.945(dd, J=8.0 Hz and 4.5 Hz, 1H, Ar—H), 6.859 (dd, J=8.0 Hz and 1.5 Hz, 1H,Ar—H), 6.739 (d, J=1.5 Hz, 1H, Ar—H), 4.192 (s, 1H, NH), 4.162 (t, J=5.0Hz, 2H, CH₂), 4.063 (br, 4H, 2 CH₂), 3.882 (t, J=5.0 Hz, 2H, CH₂),3.756-7.733 (m, 2H, CH₂), 3.705-3.624 (m, 16H, 8 CH₂), 3.592-3.509 (m,3H, CH₂ and CH), 3.370 (s, 3H, CH₃), 3.178 (t, J=5.0 Hz, 4H, 2 CH₂),1.267 (d, J=6.5 Hz, 6H, 2 CH₃). LC-MS: 658.4 (MH⁺).

Synthesis of 5-O-mPEG₇-Delavirdine 12 (n=7)

5-O-mPEG₇-indole 2-carboxylic acid 11 (n=7) (627.7 mg, 1.26 mmol) and2-piperazinyl-3-(isopropylamino)pyridine 8 (281.8 mg, 1.28 mmol) weredissolved in anhydrous THF (12 mL) at room temperature.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.40 mL, 2.19mmol) was added. The resulting mixture was stirred at room temperaturefor 6 h. 5% aq. NaHCO₃ solution was added to quench the reaction. Themixture was extracted with DCM (4×25 mL). The organic solution waswashed with brine, dried over Na₂SO₄, concentrated. The residue waspurified by flash column chromatography on SiO₂ to result in the product(574.5 mg, yield: 65%) as oil. ¹H-NMR (CDCl₃): 9.010 (br, 1H, NH), 7.693(dd, J=4.5 Hz and 1.5 Hz, 1H, Ar—H), 7.319 (d, J=9.0 Hz, 1H, Ar—H),7.059 (d, J=2.0 Hz, 1H, Ar—H), 6.993 (dd, J=9.0 Hz and 2.5 Hz, 1H,Ar—H), 6.940 (dd, J=9.0 Hz and 5.0 Hz, 1H, Ar—H), 6.854 (dd, J=8.0 Hzand 1.0 Hz, 1H, Ar—H), 6.739 (d, J=1.5 Hz, 1H, Ar—H), 4.191 (s, 1H, NH),4.163 (t, J=5.0 Hz, 2H, CH₂), 4.064 (br, 4H, 2 CH₂), 3.882 (t, J=5.0 Hz,2H, CH₂), 3.756-7.733 (m, 2H, CH₂), 3.705-3.626 (m, 20H, 10 CH₂),3.607-3.532 (m, 3H, CH₂ and CH), 3.371 (s, 3H, CH₃), 3.171 (t, J=5.0 Hz,4H, 2 CH₂), 1.266 (d, J=6.0 Hz, 6H, 2 CH₃). LC-MS: 702.4 (MH⁺).

Synthesis of 5-O-mPEG₈-Delavirdine 12 (n=8)

5-O-mPEG₈-indole 2-carboxylic acid 11 (n=8) (741 mg, 1.36 mmol) and2-piperazinyl-3-(isopropylamino)pyridine 8 (300 mg, 1.36 mmol) weredissolved in anhydrous THF (15 mL) at room temperature.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.40 mL, 2.19mmol) was added. The resulting mixture was stirred at room temperaturefor 19 h. 5% aq. NaHCO₃ solution was added to quench the reaction. Themixture was extracted with DCM (4×25 mL). The organic solution waswashed with brine, dried over Na₂SO₄, concentrated. The residue waspurified by flash column chromatography on SiO₂ to result in the product(623.3 mg, yield: 61%) as oil. ¹H-NMR (CDCl₃): 9.021 (br, 1H, NH), 7.693(dd, J=5.0 Hz and 1.5 Hz, 1H, Ar—H), 7.320 (d, J=9.0 Hz, 1H, Ar—H),7.059 (d, J=2.0 Hz, 1H, Ar—H), 6.983 (dd, J=9.0 Hz and 2.5 Hz, 1H,Ar—H), 6.940 (dd, J=9.0 Hz and 5.0 Hz, 1H, Ar—H), 6.857 (dd, J=8.0 Hzand 1.0 Hz, 1H, Ar—H), 6.739 (d, J=1.5 Hz, 1H, Ar—H), 4.191 (s, 1H, NH),4.163 (t, J=5.0 Hz, 2H, CH₂), 4.060 (br, 4H, 2 CH₂), 3.883 (t, J=5.0 Hz,2H, CH₂), 3.757-7.733 (m, 2H, CH₂), 3.700-3.627 (m, 24H, 12 CH₂),3.594-3.531 (m, 3H, CH₂ and CH), 3.371 (s, 3H, CH₃), 3.171 (t, J=5.0 Hz,4H, 2 CH₂), 1.266 (d, J=6.5 Hz, 6H, 2 CH₃). LC-MS: 746.5 (MH⁺).

Synthesis of 5-O-mPEG₉-Delavirdine 12 (n=9)

5-O-mPEG₉-indole 2-carboxylic acid 11 (n=9) (952 mg, 1.62 mmol) and2-piperazinyl-3-(isopropylamino)pyridine 8 (356 mg, 1.62 mmol) weredissolved in anhydrous THF (15 mL) at room temperature.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.50 mL, 2.19mmol) was added. The resulting mixture was stirred at room temperaturefor 18 h. 5% aq. NaHCO₃ solution was added to quench the reaction. Themixture was extracted with DCM (4×25 mL). The organic solution waswashed with brine, dried over Na₂SO₄, concentrated. The residue waspurified by flash column chromatography on SiO₂ to result in the product(779.2 mg, yield: 61%) as oil. ¹H-NMR (CDCl₃): 9.057 (br, 1H, NH), 7.697(dd, J=4.5 Hz and 1.5 Hz, 1H, Ar—H), 7.325 (d, J=9.0 Hz, 1H, Ar—H),7.062 (d, J=2.0 Hz, 1H, Ar—H), 6.996 (dd, J=9.0 Hz and 2.5 Hz, 1H,Ar—H), 6.944 (dd, J=8.0 Hz and 5.0 Hz, 1H, Ar—H), 6.858 (dd, J=8.0 Hzand 1.0 Hz, 1H, Ar—H), 6.742 (d, J=1.5 Hz, 1H, Ar—H), 4.196 (s, 1H, NH),4.167 (t, J=5.0 Hz, 2H, CH₂), 4.065 (br, 4H, 2 CH₂), 3.886 (t, J=5.0 Hz,2H, CH₂), 3.761-7.742 (m, 2H, CH₂), 3.704-3.635 (m, 28H, 14 CH₂),3.612-3.535 (m, 3H, CH₂ and CH), 3.376 (s, 3H, CH₃), 3.175 (t, J=5.0 Hz,4H, 2 CH₂), 1.270 (d, J=6.0 Hz, 6H, 2 CH₃). LC-MS: 702.4 (MH⁺)

Example 2 Synthesis of mPEG_(n)-N-Delavirdine Conjugates

Synthesis of Ethylpyruvate 4-Nitrophenylhydrazone 14

Glacial acetic acid (3.0 mL, 48.38 mmol) was added to a stirred solutionof 4-nitrophenylhydrazine (from Aldrich, >30% water as stabilizer, 9.0g, 39.9 mmol) in ethyl alcohol (350 mL) at room temperature. And ethylpyruvate (7.2 mL, 61.68 mmol) was added. The resulting mixture wasstirred at room temperature for one hour and at 60° C. for 3.5 h, cooledto room temperature. The mixture was adjusted to pH about 10.25 with aqKOH solution. The mixture was concentrated to remove organic solvents.The brown solid was collected by filtering and washing with water toafford the product (8.19 g) in 82% yield. The product was direct usedfor the next step without purification. ¹H-NMR (CDCl₃): 8.217 (d, J=9.0Hz, 2H, Ar—H), 7.27 (d, J=9.0 Hz, 2H, Ar—H), 4.348 (q, J=7.0 Hz, 2H,CH₂), 2.173 (s, 3H, CH₃), 1.399 (t, J=7.0 Hz, 3H, CH₃). (From the¹H-NMR, there are about 10% of another isomer).

Synthesis of 5-Nitro-1H-indole-2-carboxylic Acid Ethyl Ester 15

Ethylpyruvate 4-nitrophenylhydrazone 14 (8.18 g, 32.56 mmol) was heatedat 125° C. in polyphosphoric acid (80 g) for 5 h. The mixture was cooledto room temperature, poured into water containing some ice. The solidwas filtered through a funnel, washed with water. The solid wascollected and recrystallized with ethyl acetate to afford the product(5.47 g, 72% yield). ¹H-NMR (DMSO-d₆): 8.734 (d, J=2.0 Hz, 1H, Ar—H),8.131 (dd, J=2.0 Hz and 9.0-9.5 Hz, 1H, Ar—H), 7.606 (d, J=9.0-9.5 Hz,1H, Ar—H), 7.442 (br, 1H, Ar—H), 4.378 (q, J=7.0-7.5 Hz, 2H, CH₂), 1.355(t, J=7.0-7.5 Hz, 3H, CH₃).

Synthesis of 5-Nitro-1H-indole-2-carboxylic Acid 16

5-Nitro-1H-indole-2-carboxylic acid ethyl ester 15 (2.73 g, 11.65 mmol)and KOH (2.69 g, 41.33 mmol) were stirred in dioxane/water (50 mL/10 mL)at room temperature for 41 h. Water was added to diluted the solution.The mixture was concentrated to remove the organic solvent. Theremaining solution was washed with DCM (100 mL). The aqueous solutionwas adjusted with 1 N HCl to pH 4.16, extracted with EtOAc (3×100 mL).The combined EtOAc extraction was washed with brine, dried over sodiumsulfate, concentrated to afford the product as solid (1.79 g, 74%yield).

5-Nitro-1H-indole-2-carboxylic acid ethyl ester 15 (194 mg, 0.83 mmol)and KOH (342 mg, 5.25 mmol) were stirred in MeOH/water (8 mL/4 mL) atroom temperature for 21.5 h. More water was added to dilute thesolution. The mixture was concentrated to remove the organic solvent.The remaining solution was washed with DCM (100 mL). The aqueoussolution was adjusted with 1 N HCl to pH 2.9, extracted with EtOAc (2×15mL). The combined EtOAc extraction was washed with brine, dried oversodium sulfate, concentrated to afford the product as solid (185.5 mg,97% yield). ¹H-NMR (DMSO-d₆): 8.718 (d, J=2.0-2.5 Hz, 1H, Ar—H), 8.116(dd, J=2.0-2.5 Hz and 9.0-9.5 Hz, 1H, Ar—H), 7.583 (d, J=9.0-9.5 Hz, 1H,Ar—H), 7.378 (d, J=1.5 Hz, 1H, Ar—H). LC-MS: 207.1 (MH⁺).

Synthesis of 5-Nitro-Delavirdine 17

EDC (1.75 mL, 9.59 mmol) was added to a stirred solution of 5-nitroindole acid (1.65 g, 8.0 mmol) and3-isopropylamino-2-piperazinyl-pyridine (1.951 g, 8.86 mmol) in THF (75mL) at room temperature. The resulting mixture was stirred at roomtemperature for 26.5 h. 5% NaHCO₃ aq solution (100 mL) was added toquench the reaction. The mixture was concentrated to remove the organicsolvent. The remaining mixture was extracted with DCM (4×100 mL). Thecombined organic solution was concentrated to afford a residue. Theresidue was mixed with small amount of DCM (˜15 mL), filtered and washedthe solid with DCM to afford a solid as product. The above aqueoussolution (after extraction) was filtered through a Celite funnel andwashed the solid with acetone and DCM, dried to afford another part ofproduct. The total yield was 50%. ¹H-NMR (CDCl₃): 9.640 (s, 1H, NH),8.640 (d, J=2.5 Hz, 1H, Ar—H), 8.184 (dd, J=2.5 Hz and 9.0-9.5 Hz, 1H,Ar—H), 7.700 (dd, J=1.5 and 5.0 Hz, 1H, Ar—H), 7.489 (d, J=9.0-9.5 Hz,1H, Ar—H), 6.988-6.947 (m, 2H, 2 Ar—H), 6.883-6.867 (m, 1H, Ar—H),4.189-4.090 (m, 5H, NH and 2 CH₂), 3.618-3.554 (m, 1H, CH), 3.204 (m,4H, 2 CH₂), 1.275 (d, J=6.0 Hz, 6H, 2 CH₃). LC-MS: 409.2 (MH⁺).

Synthesis of 5-Amino-Delaviridine 18

A mixture of 5-nitro delavirdine 17 (1.1773 g, 2.88 mmol) andcyclohexene (2.0 mL, 19.52 mmol) in THF/MeOH (80/100 mL) in the presenceof Pd(OH)₂ on activated carbon (Pearlman's catalyst) (2.74 g) wasstirred at 75° C. for 7.5 h. The reaction mixture was cooled to roomtemperature. The mixture was filtered through a pad of Celite. TheCelite and the palladium were washed with MeOH (2×15 mL). The combinedsolution was concentrated. The residue was purified by flash columnchromatography on silica gel, eluting with 50-100% EtOAc/hexane toafford 0.5974 g of 5-amino delavirdine 18. ¹H-NMR (CDCl₃): 8.857 (s, 1H,NH), 7.692 (dd, J=1.5 and 5.0 Hz, 1H, Ar—H), 7.242 (d, J=8.5-9.0 Hz, 1H,Ar—H), 6.950-6.901 (m, 2H, 2 Ar—H), 6.860-6.841 (m, 1H, Ar—H), 6.774(dd, J=2.0-2.5 and 8.5-9.0 Hz, 1H, 1 Ar—H), 6.642 (d, J=1.5 Hz, 1H,Ar—H), 4.181 (m, 1H, NH), 4.069 (m, 2 CH₂), 3.576 (m, 3H, NH₂ and CH),3.164 (m, 4H, 2 CH₂), 1.264 (d, J=6.5 Hz, 6H, 2 CH₃). LC-MS: 379.2(MH⁺).

Synthesis of 5-mPEG-NH-Delavirdine 19 (n=1)

A reaction mixture of 5-amino delavirdine (390 mg, 1.03 mmol), mPEG₁-OMs(183.8 mg, 1.19 mmol), potassium carbonate (791 mg, 5.72 mmol) andtetrabutylammonium bromide (33 mg, 0.102 mmol) in CH₃CN/DMF (9/2 mL) wasplaced in single-mode focused microwave reactor (CEM) and irradiated at120° C. for 150 min. The mixture was concentrated to remove the organicsolvent and the residue was mixture with dichloromethane (120 mL),washed with water (2×80 mL), dried over Na₂SO₄, and concentrated. Theresidue was purified by column chromatography to afford the product pure5-mPEG₁-NH-delavirdine 18 (n=1) (103.2 mg), as well as some startingmaterial (89.5 mg). ¹H-NMR (CDCl₃): 9.568 (s, 1H, NH), 7.691 (dd,J=1.0-1.5 and 4.5-5.0 Hz, 1H, Ar—H), 7.237 (d, J=9.0 Hz, 1H, Ar—H),6.941-6.916 (m, 1H, Ar—H), 6.851-6.835 (m, 1H, Ar—H), 6.787 (m, 1H,Ar—H), 6.738 (dd, J=2.0 and 9.0 Hz, 1H, 1 Ar—H), 6.662 (d, J=1.0-1.5 Hz,1H, Ar—H), 4.190 (d, J=7.0 Hz, 1H, NH), 4.067 (m, 4H, 2 CH₂), 3.640 (t,J=5.0 Hz, CH₂), 3.593-3.529 (m, 1H, CH), 3.391 (s, 3H, CH₃), 3.308 (t,J=5.0 Hz, 2H, CH₂), 3.196 (t, J=5.0 Hz, 4H, 2 CH₂), 1.255 (d, J=6.0 Hz,6H, 2 CH₃). LC-MS: 437.3 (MH⁺).

Synthesis of 5-mPEG₂-NH-Delavirdine 19 (n=2)

A reaction mixture of 5-amino delavirdine (244.6 mg, 0.647 mmol),mPEG₂-OMs (176.7 mg, 0.891 mmol), potassium carbonate (514.8 mg, 3.725mmol) and tetrabutylammonium bromide (56 mg, 0.174 mmol) in CH₃CN/DMF(3.0/1.9 mL) was placed in single-mode focused microwave reactor (CEM)and irradiated at 120° C. for 150 min. The mixture was concentrated toremove the organic solvent and the residue was mixture with water (15mL), extracted with dichloromethane (3×40 mL). The combined organicsolution was washed with brine, dried over Na₂SO₄, concentrated. Theresidue was purified by column chromatography to afford the product pure5-mPEG₂-NH-delavirdine 18 (n=2) (50.4 mg), as well as some startingmaterial and 5-di-mPEG₂-H-delavirdine. ¹H-NMR (CDCl₃): 9.450 (s, 1H,NH), 7.719 (dd, J=1.5 and 5.0 Hz, 1H, Ar—H), 7.266 (d, J=8.5-9.0 Hz, 1H,Ar—H), 6.970-6.945 (m, 1H, Ar—H), 6.881-6.862 (m, 1H, Ar—H), 6.813 (m,1H, Ar—H), 6.768 (dd, J=2.0-2.5 and 8.5-9.0 Hz, 1H, 1 Ar—H), 6.688 (d,J=1.5 Hz, 1H, Ar—H), 4.216 (d, J=7.0 Hz, 1H, NH), 4.092 (m, 4H, 2 CH₂),3.769 (t, J=5.0-5.5 Hz, CH₂), 3.685-3.667 (m, 2H, CH₂), 3.611-3.562 (m,4H, CH, NH and CH₂), 3.423 (s, 3H, CH₃), 3.356 (t, J=5.0-5.5 Hz, 2H,CH₂), 3.196 (m, 4H, 2 CH₂), 1.285 (d, J=6.0 Hz, 6H, 2 CH₃). LC-MS: 481.5(MH⁺).

Synthesis of 5-mPEG₃-NH-Delavirdine 19 (n=3)

A reaction mixture of 5-amino delavirdine (100.2 mg, 0.265 mmol),mPEG₃-OMs (83.8 mg, 0.346 mmol), potassium carbonate (198.4 mg, 1.435mmol) and tetrabutylammonium bromide (15 mg, 0.174 mmol) in CH₃CN (1.5mL) was placed in single-mode focused microwave reactor (CEM) andirradiated at 120° C. for 110 min. The mixture was cooled to roomtemperature and filtered. The solid was washed with acetonitrile. Thecombined organic solution was concentrated to remove the organicsolvent. The residue was purified by column chromatography to afford theproduct pure 5-mPEG₃-NH-delavirdine 18 (n=3) (31.7 mg), as well as somestarting material. ¹H-NMR (CDCl₃): 9.219 (s, 1H, NH), 7.690 (dd, J=1.5and 5.0 Hz, 1H, Ar—H), 7.239 (d, J=8.5-9.0 Hz, 1H, Ar—H), 6.943-6.918(m, 1H, Ar—H), 6.855 6.836 (m, 1H, Ar—H), 6.782 (m, 1H, Ar—H), 6.751(dd, J=2.0-2.5 and 8.5-9.0 Hz, 1H, 1 Ar—H), 6.661 (d, J=1.5 Hz, 1H,Ar—H), 4.182 (br, 1H, NH), 4.059 (m, 4H, 2 CH₂), 3.740 (t, J=5.0 Hz,CH₂), 3.670-3.646 (m, 7H, NH and 3 CH₂), 3.565-3.547 (m, 3H, CH andCH₂), 3.379 (s, 3H, CH₃), 3.318 (t, J=5.0 Hz, 2H, CH₂), 3.166 (m, 4H, 2CH₂), 1.259 (d, J=6.0 Hz, 6H, 2 CH₃). LC-MS: 525.3 (MH+).

Synthesis of Ethyl 5-Amino-1H-indole 2-Carboxylate 20

A mixture of 5-Nitro-1H-indole-2-carboxylic acid ethyl ester 15 (88.7mg, 0.38 mmol) and cyclohexene (0.1 mL, 0.98 mmol) in THF/MeOH (3/8 mL)in the presence of Pd(OH)₂ on activated carbon (Pearlman's catalyst)(2.74 g) was heated to reflux for 5 h. The reaction mixture was cooledto room temperature. The mixture was filtered through a pad of Celite.The Celite and the palladium were washed with EtOAc. The combinedsolution was concentrated. The residue was purified by flash columnchromatography on silica gel, eluting with 5-45% EtOAc/hexane to afford66.6 mg of ethyl 5-amino-1H-indole 2-carboxylate 20. ¹H-NMR (CDCl₃):8.653 (Br, 1H, NH), 7.228 (d, J=8.5-9.0 Hz, 1H, Ar—H), 7.031 (dd, J=1.0Hz and 2.0 Hz, 1H, Ar—H), 6.932 (d, J=2.5 Hz, 1H, Ar—H), 6.803 (dd,J=2.5 Hz, 1H, Ar—H), 4.379 (q, J=7.0-7.5 Hz, 2H, CH₂), 1.396 (t,J=7.0-7.5 Hz, 3H, CH₃). LC-MS: 205.2 (MH⁺).

Synthesis of 5-mPEG₃-amino 1H-indole 2-Carboxylic Acid 21

A reaction mixture of ethyl 5-amino-1H-indole 2-carboxylate 20 (36.3 mg,0.18 mmol), mPEG₃-OMs (65.2 mg, 0.27 mmol), and potassium carbonate (60mg, 0.434 mmol) in water (1 mL) was placed in single-mode focusedmicrowave reactor (CEM) and irradiated at 120° C. for 30 min. Themixture was cooled to room temperature and adjusted the pH to 4.03 with1N HCl, washed with dichloromethane, extracted with EtOAc (3×15 mL). Thecombined ethyl acetate solution was washed with brine, dried overNa2SO4, concentrated to afford the product as the major one based on theresults of ¹H-NMR in MeOD and LC-MS.

Example 3 pKa and Log P Determination for Various Conjugates

The Sirius GLpKa instrument is used to determine the pKa and Log P. Ablank standardization has to be completed first and passed in order toconduct the compound experiments. The solutions used by the instrumentfor the blank titration include water, a pH solution, and a surfactant(TRITON X-100). The TRITON X-100 was purchased commercially from Sirus.The 0.5% solution of TRITON X-100 was used in the experiment. In a 1 Lvolumetric flask 5 mls of TRITON X-100 is added and MILLI Q water tovolume. The vessels used by the instrument were filled to ⅓ of theirvolume with each solution. Therefore the assay tray contains one vesselfor water, pH solution, and surfactant for the blank run. Thetemperature of the water bath for the instrument is 25° C. The blank iscalculated as an average of three “good” experiments. The “good” wasdenoted by a green check mark next to the assay number in the computersoftware. Once the blank was run successfully the instrument was readyto conduct the experiments.

The pKa has to be determined first in order to calculate the Log P. TheAPI is used to determine the pKa needed for the Log P experimentfollowed by the conjugates. The base compound was first weighed into avessel. The amount weighed for each sample was 10% of the molecularweight of the compound. The same procedure was followed for eachconjugate. The vessels were placed in the assay tray of the instrument.A method had to be set up for the pKa run. The experimental parametersare entered into the computer software. These included sample weight,molecular weight, assay type (aqueous pKa), number of assays (three),and titration range (generally 3.5-12). The run was started. There arethree measurements for each run that are averaged to obtain the finalresults from the data points collected in the calculation software. Allruns were denoted as “good” by the green check mark next to the assaynumber. The pKa values were obtained for all compounds as listed in thetable below.

An additional method has to be set up for the Log P determinations foreach compound. The conjugates are weighed into vessels in the samemanner as for the previous experiment, i.e., 10% of the molecularweight. The vessels are placed in the assay tray of the instrument. Theexperimental parameters are entered into the computer. These includedsample weight, molecular weight, assay type (Partition Log P), number ofassays (three), and pKa value of the parent drug from the previousexperiment. The run was started. There are three measurements for eachrun that are averaged to obtain the final results from the data pointscollected in the calculation software. All runs were denoted as “good”by the green check mark next to the assay number. The Log P values wereobtained for all compounds as listed in Table 1 below.

TABLE 1 pK and Log P Values of Tested Compounds Compound pKa1 pKa2 Log PDelavirdine Mesylate 4.323 9.272 3.5 mPEG3-O-Delavirdine 3.792 1.981mPEG4-O-Delavirdine 4.275 2.217 mPEG5-O-Delavirdine 4.454mPEG6-O-Delavirdine 4.447 2.645 mPEG7-O-Delavirdine 4.683 2.733mPEG8-O-Delavirdine 4.667 2.578 mPEG9-O-Delavirdine 4.655 2.328

Example 4 Anti-HIV-1 Cytoprotection Assay

Cell Preparation—CEM-SS cells were passaged in T-75 flasks prior to usein the antiviral assay. On the day preceding the assay, the cells weresplit 1:2 to assure they were in an exponential growth phase at the timeof infection. Total cell and viability quantification was performedusing a hemocytometer and Trypan Blue dye exclusion. Cell viability wasgreater than 95% for the cells to be utilized in the assay. The cellswere resuspended at 5×10⁴ cells per ml in tissue culture medium andadded to the drug containing microtiter plates in a volume of 50 μL.

Virus Preparation—The virus used for the assay was the lymphocyte-tropicvirus strain HIV-I_(RF). The virus was obtained from the NIH AIDSResearch and Reference Reagent Program and stock virus pools wereproduced in CEM-SS cells. A pretitred aliquot of virus was removed fromthe freezer (−80° C.) and allowed to thaw slowly to room temperature ina biological safety cabinet. Virus was re-suspended and diluted intotissue culture medium such that the amount of virus added to each wellin a volume of 50 μL was the amount determined to yield 85 to 95% cellkilling at 6 days post-infection.

Plate Format—Each plate contains cell control wells (cells only), viruscontrol wells (cells plus virus), drug toxicity wells (cells plus drugonly), drug colorimetric control wells (drug only) as well asexperimental wells (drug plus cells plus virus). Samples were tested intriplicate with eleven half-log dilutions per compound.

Efficacy and Toxicity XTT—Following incubation at 37° C. in a 5% CO₂incubator, the test plates were stained with the tetrazolium dye XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide). XTT-tetrazolium is metabolized by the mitochondrial enzymesof metabolically active cells to a soluble formazan product, allowingrapid quantitative analysis of the inhibition of HIV induced cellkilling by anti-HIV test substances. XTT solution was prepared daily asa stock of 1 mg/mL in RPMI1640. Phenazine methosulfate (PMS) solutionwas prepared at 0.15 mg/mL in PBS and stored in the dark at −20° C.XTT/PMS stock was prepared immediately before use by adding 40 μL of PMSper ml of XTT solution. Fifty microliters of XTT/PMS was added to eachwell of the plate and the plate was re-incubated for 4 hours at 37° C.Plates were sealed with adhesive plate sealers and shaken gently orinverted several times to mix the soluble fosmazan product and the platewas read spectrophotometrically at 450/650 nm with a Molecular DevicesVmax plate reader.

Example 5 HIV-1 Reverse Transcriptase Inhibition Assay

The following materials were removed from the freezer and were stored onwet ice (dGTP, ³²P, purified enzyme extract, rCdG, dGTP, DNA). Testcompounds were evaluated at a high test of 100 μM diluted in dH₂O and ifthe stock solution was turbid, 1:1000 Triton-X was added to the stocksolution. Thirty microliters (30 μL) of each dilution was thentransferred in duplicate to the appropriate well of a 96-well plate. Thepositive and negative control wells each received 30 μL of dH₂O insteadof the compound. A 2× buffer was prepared in the following manner: 125μL 2M Tris pH 8.0, 250 μL 3M KCl, 80 μL 1 M MgC12, 10 μL 2 M DTT, 40 μL25 U/mL rCdG, 100 μL 1 mM dGTP, 10 μL 800 Ci/mMol alpha ³²P:dG TP, and4.4 mL dH₂O. Fifty microliters (50 μL) of 2× buffer was added to everywell of the plate. BSA was diluted 1:100 and Triton-X 1:1000 in dH₂O andthe enzyme was added to the mixture at a 1:400 dilution. Twentymicroliters (20 μL) of the enzyme solution was added to all of the wellsof the plate except the negative control wells. Distilled water was usedinstead of enzyme in these wells. The plate was incubated at 37° C. for30 minutes. Following incubation, 10 μL of 10 mg/mL DNA (MB-grade fishsperm) was added to each well, followed by 150 μL of cold 10% TCA ineach well. The plate was incubated at room temperature for approximately15 minutes in order to allow for precipitation of the samples. Followingthe incubation the contents were transferred to the appropriate filterplate. The plate was placed on the vacuum manifold and pushed firmly toinduce suction. Wells were rinsed with 200 μL of cold TCA. Again, theplate was pushed down firmly and vacuum suction was applied. Once all ofthe liquid was aspirated from the filter plate, it was removed from themanifold and residual liquid was blotted from the bottom of the plasticbase. The filtered wells were separated from the plastic base and thewells were blotted on clean, absorbent paper. The filtered wells werepushed into the cassette and a sheet of Millipore multiscreen tape wasplaced on the bottom of a Wallace Microbeta cassette covering all of theholes. Twenty microliters (20 μL) of Wallace Supermix Scintillant wasadded to each well and the top of the wells were covered with a sheet ofthe multiscreen tape. The plate was read on the Wallace MicroBetacounter to measure incorporated ³²P.

Anti-HIV-1 Cytopathic Effects Inhibition Assay Evaluations: Eightcompounds were evaluated against the RF strain of HIV-1 in CEM-SS cellsusing a 12 concentration dose response curve. The results of theseassays are summarized in the table below. The AZT control compound wasevaluated in parallel with the test materials and yielded an EC₅₀ valueof 6.0 nM, which falls within the acceptable range of activity of thecontrol compound (1 to 10 nM).

Example 6 Anti-HIV Evaluation of Conjugates in CEM-SS Cells Against WildType and Drug Resistant HIV-RF

Cells were prepared as described above. Virus Preparation—The wild typeNL4-3 and drug resistant A17 viruses were obtained from the NIH AIDSResearch and Reference Reagent Program and stock virus pools wereproduced in CEM-SS cells. Amino acid substitutes of L100I, Y181C andP23L were introduced by site-directed mutagenesis into pN L4-3 and stockvirus pools were produced in CEM-SS cells. A pretitered aliquot of viruswas removed from the freezer (−80° C.) and allowed to thaw slowly toroom temperature in a biological safety cabinet. Virus was re-suspendedand diluted into tissue culture medium such that the amount of virusadded to each well in a volume of 50 μL was the amount determined toyield 85 to 95% cell killing at 6 days post-infection.

Plate Format—Each plate contains cell control wells (cells only), viruscontrol wells (cells plus virus), drug toxicity wells (cells plus drugonly), drug colorimetric control wells (drug only) as well asexperimental wells (drug plus cells plus virus). Samples were tested intriplicate with eleven half-log dilutions per compound. Efficacy andToxicity XTT—Following incubation at 37° C. in a 5% CO₂ incubator, thetest plates were stained with the tetrazolium dye XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide). XTT-tetrazolium was metabolized by the mitochondrial enzymesof metabolically active cells to a soluble formazan product, allowingrapid quantitative analysis of the inhibition of HIV induced cellkilling by anti-HIV test substances. XTT solution was prepared daily asa stock of 1 mg/mL in RPMI1640. Phenazine methosulfate (PMS) solutionwas prepared at 0.15 mg/mL in PBS and stored in the dark at −20° C.XTTIPMS stock was prepared immediately before use by adding 40 μL of PMSper ml of XTT solution. Fifty microliters of XTT/PMS was added to eachwell of the plate and the plate was re-incubated for 4 hours at 37° C.Plates were sealed with adhesive plate sealers and shaken gently orinverted several times to mix the soluble formazan product and the platewas read spectrophotometrically at 450/650 nm with a Molecular DevicesVmax plate reader.

Data Analysis—Raw data was collected from the Softmax Pro 4.6 softwareand imported into a Excel XLFit4 spreadsheet for analysis by fourparameter curve fit calculations. Both antiviral activity and toxicityare calculated. The results of these assays are summarized in the tablebelow.

Anti-HIV Activities and Cytotoxicities

CEM-SS/ CEM- Wild HIV-1_(RF) SS Type RT EC₅₀ TC₅₀ Therapeutic CompoundIC₅₀ (μM) (μM) (μM) Index AZT 0.006 >1.0 >166.7 (Repeat test)0.01 >1.0 >100.0 Delavirdine Mesylate 0.027 0.002 27.2 13587.0 (Repeattest) 0.001 0.006 52.43 8738.33 mPEG3-O-Delavirdine 0.05 0.054 26.9499.0 mPEG4-O-Delavirdine 0.28 0.16 34.0 213.8 mPEG5-O-Delavirdine 0.130.76 14.4 19.0 mPEG6-O-Delavirdine 0.29 0.84 22.5 26.7mPEG7-O-Delavirdine 0.70 0.47 35.5 76.1 mPEG8-O-Delavirdine 0.49 0.3652.5 144.2 mPEG9-O-Delavirdine 0.56 0.13 40.7 323.1 5-mPEG1-NH- 0.250.047 50.91 1083.11 Delavirdine 5-mPEG2-NH- 0.58 0.017-~0.2* 55.533266.29 Delavirdine 5-mPEG3-NH- 1.0 0.16 34.88 212.67 Delavirdine*Note - variability in cytoprotection data at 0.032 and 0.102 μM made itdifficult to accurately determine the EC₅₀ value. Ignoring thevariability, the EC₅₀ was calculated as 0.017 μM.

Anti-HIV Cytoprotection Assay Using Drug Resistant Virus Strains

AZT Nevirapine Delavirdine EC₅₀ TC₅₀ EC₅₀ TC₅₀ EC₅₀ TC₅₀ HIV-1 Strain(μM) (μM) TI (μM) (μM) TI (μM) (μM) TI NL4-3 0.004 >0.5 >125.00.19 >10.0 >52.6 0.005 >0.5 >100.0 (repeat test) 0.01 >0.1 >10.00.09 >10.0 >111.0 0.01 >1.0 >100.0 NL4-3_(L100I)0.003 >0.5 >167.0 >10.0 >10.0 — 0.003 >0.5 >167.0 (repeat test)0.006 >0.1 >16.7 >10.0 >10.0 — 0.24 >1.0 >4.2 NL4-3_(Y181C)0.002 >0.5 >250.0 >10.0 >10.0 — >0.5 >0.5 — (repeat test)0.001 >0.1 >100.0 8.59 >10.0 >1.2 0.58 >1.0 >1.7 NL4-3_(P236L)0.007 >0.5 >71.4 0.3 >10.0 >33.3 >0.5 >0.5 — (repeat test)0.002 >0.1 >50.0 0.28 >10.0 >35.7 0.5 >1.0 >2.0 A17_(K103N/Y181C)0.003 >0.5 >167.0 >10.0 >10.0 — >0.5 >0.5 — (repeat test)0.003 >0.5 >167.0 >10.0 >10.0 — >1.0 >1.0 — mPEG₃-O-delavirdinemPEG₄-O-delavirdine mPEG₉-O-delavirdine EC₅₀ TC₅₀ EC₅₀ TC₅₀ EC₅₀ TC₅₀HIV-1 Strain (μM) (μM) TI (μM) (μM) TI (μM) (μM) TI NL4-30.06 >5.0 >83.3 0.87 >5.0 >5.8 0.89 >5.0 >5.6 NL4-3_(L100I)0.03 >5.0 >167.0 0.95 >5.0 >5.3 0.94 >5.0 >5.3 NL4-3_(Y181C) >5.0 >5.0— >5.0 >5.0 — >5.0 >5.0 — NL4-3_(P236L) >5.0 >5.0 — >5.0 >5.0— >5.0 >5.0 — A17_(K103N/Y181C) >5.0 >5.0 — >5.0 >5.0 — >5.0 >5.0 —5-mPEG₁-NH-delavirdine 5-mPEG₂-NH-delavirdine EC₅₀ TC₅₀ EC₅₀ TC ₅₀ HIV-1Strain (μM) (μM) TI (μM) (μM) TI NL4-3 0.02 >1.0 >50.0 0.08 >1.0 >12.5NL4-3_(L100I) 0.56 >1.0 >1.8 0.71 >1.0 >1.4 NL4-3_(Y181C)0.82 >1.0 >1.2 >1.0 >1.0 — NL4-3_(P236L) >1.0 >1.0 — 0.23 >1.0 >4.4A17_(K103N/Y181C) >1.0 >1.0 — >1.0 >1.0 —

Example 7 Caco-2 Permeability Studies

The objective of this study was to determine the bidirectional Caco-2permeability of test compounds. Caco-2 monolayers were grown toconfluence on collagen-coated, microporous, polycarbonate membranes in12-well Costar Transwell® plates. Details of the plates and theircertification are shown below. The permeability assay buffer for thedonor chambers was Hanks Balanced Salt Solution containing 10 mM HEPESand 15 mM glucose (HBSSg) at a pH of 7.4. The buffer in the receiverchambers also contained 1% bovine serum albumin (BSA). The dosingsolution concentrations were 10 μM in the assay buffer. Cell monolayerswere dosed on the apical side (A to-B) or basolateral side (B-to-A) andincubated at 37° C. with 5% CO₂ in a humidified incubator. After 2hours, aliquots were taken from the receiver and donor chambers. Eachdetermination was performed in duplicate. The lucifer yellow flux wasalso measured for each monolayer to ensure no damage was inflicted tothe cell monolayers during the flux period. All samples were assayed byLC/MS/MS using electrospray ionization. Analytical conditions areoutlined in Appendix A. The apparent permeability, P_(app), and percentrecovery were calculated as follows:P _(app)=(dC _(r) /dt)×V _(r)/(A×C _(A))  (1)Percent Recovery=100×((V _(r) ×C _(r) ^(final))+(V _(d) ×C _(d)^(final)))/(V _(d) ×C _(N))  (2)where, dCr/dt is the slope of the cumulative concentration in thereceiver compartment versus time in μM s-1.

V_(r) is the volume of the receiver compartment in cm³.

V_(d) is the volume of the donor compartment in cm³.

A is the area of the cell monolayer (1.13 cm² for 12-well Transwell®).

C_(N) is the nominal dosing concentration of the dosing solution in μM.

C_(A) is the average of the nominal dosing concentration and themeasured 120 minute donor concentration in μM.

C_(r) ^(final) is the receiver concentration in μM at the end of theincubation period.

C_(d) ^(final) is the concentration of the donor in μM at the end of theincubation period.

Description of Caco-2 Permeability Characterization (Test 1)

Passage Number 65 Acceptance Criteria Age at QC (Days) 23 TEER Value (Ω· cm²) 563 450-650 Lucifer Yellow P_(app), × 10⁻⁶ cm/s 0.18 <0.4Atenolol P_(app), × 10⁻⁶ cm/s 0.26 <0.5 Propranolol P_(app), × 10⁻⁶ cm/s20.8 15-25 Digoxin (A-to-B) P_(app,) × 10⁻⁶ cm/s 0.47 N/A Digoxin(B-to-A) P_(app,) × 10⁻⁶ cm/s 11.0 N/A Digoxin Efflux Ratio 23 >3.0

Recovery and Apparent Permeability (Test 1)

Average Efflux Average P_(app), P_(app), Ratio Recovery (%) A→B B→A(B→A/ Compound A→B B→A (×10⁻⁶ cm/s) (×10⁻⁶ cm/s) A→B) Delavirdine 65 659.87 67.9 6.9 Mesylate mPEG3-O- 66 61 38.4 33.9 0.9 Delavirdine mPEG4-O-74 65 39.3 34.8 0.9 Delavirdine mPEG5-O- 71 65 29.9 39.1 1.3 DelavirdinemPEG6-O- 67 72 16.1 34.1 2.1 Delavirdine mPEG7-O- 74 69 14.2 36.6 2.6Delavirdine mPEG8-O- 80 82 4.98 43.6 8.8 Delavirdine mPEG9-O- 66 72 2.2227.7 12 Delavirdine

Description of Caco-2 Permeability Characterization (Test 2)

Acceptance Passage Number 67 59 Criteria Age at QC (Days) 20 21 TEERValue (Ω · cm²) 600 574 450-650 Lucifer Yellow P_(app), × 10⁻⁶ cm/s 0.230.16 <0.4 Atenolol P_(app), × 10⁻⁶ cm/s 0.29 0.24 <0.5 PropranololP_(app), × 10⁻⁶ cm/s 17.0 15.4 15-25 Digoxin (A-to-B) P_(app,) × 10⁻⁶cm/s 0.75 0.65 N/A Digoxin (B-to-A) P_(app,) × 10⁻⁶ cm/s 13.4 16.9 N/ADigoxin Efflux Ratio 18 26 >10

Recovery and Apparent Permeability (Test 2)

Average Average Efflux Recovery P_(app), P_(app), Ratio (%) A→B B→A(B→A/ Compound A→B B→A (×10⁻⁶ cm/s) (×10⁻⁶ cm/s) A→B) Delavirdine 82 8616.1 56.6 3.5 Mesylate mPEG3-O- 95 101 41.4 29.1 0.7 DelavirdinemPEG5-O- 97 95 33.3 38.9 1.2 Delavirdine mPEG7-O- 94 95 23.1 39.3 1.7Delavirdine mPEG9-O- 93 94 5.27 37.3 7.1 Delavirdine 5-mPEG1-NH- 91 9250.2 39.4 0.8 Delavirdine 5-mPEG2-NH- 88 88 46.2 41.1 0.9 Delavirdine5-mPEG3-NH- 94 96 33.3 41.8 1.3 Delavirdine

What is claimed is:
 1. A method comprising administering to a subject acompound having the following structure,

wherein n is an integer between 1 and 30, inclusive, and pharmaceuticalsalts thereof.
 2. The method of claim 1, wherein n is an integer between1 and
 10. 3. The method of claim 1, wherein the compound is present in acomposition comprising a pharmaceutically acceptable excipient.
 4. Themethod of claim 1, wherein the compound is present in a dosage form. 5.The method of claim 1, wherein n is
 1. 6. The method of claim 1, whereinn is
 2. 7. The method of claim 1, wherein n is
 3. 8. The method of claim1, wherein n is
 4. 9. The method of claim 1, wherein n is
 5. 10. Themethod of claim 1, wherein n is
 6. 11. The method of claim 1, wherein nis
 7. 12. The method of claim 1, wherein n is
 8. 13. The method of claim1, wherein n is
 9. 14. The method of claim 1, wherein n is 10.